Plasma Proteomic Profile Associated with Platelet Dysfunction after Trauma

Background. Coagulopathic bleeding is a major cause of mortality after trauma, and platelet dysfunction contributes to this problem. The causes of platelet dysfunction are unknown, but a great deal can be learned from the plasma environment after injury, which may directly alter platelet function. Studying the changes in plasma using untargeted proteomics would provide unbiased insight into the presence of possible inhibitors of platelet function, changes to their major ligands, or other previously unknown pathways affecting platelet function. Methods. Citrated blood was collected from severely injured trauma patients at the time of their arrival to the Emergency Department. Platelet testing was performed immediately, and plasma was frozen for analysis. Samples were collected from 110 patients, and a subset of 24 patients was identified by a preserved (n=12) or severely impaired (n=12) platelet aggregation response to five different agonists (adenosine diphosphate, arachidonic acid, collagen, thrombin receptor-activating peptide, and ristocetin). Untargeted proteomics was performed by nanoflow liquid chromatography tandem mass spectrometry to determine the plasma protein profile associated with platelet dysfunction. Protein abundance levels for each patient were normalized to total protein concentration to control for hemodilution by crystalloid fluid infusion prior to blood draw. Results were compared by Wilcoxon rank-sum test, and a two-tailed p value less than 0.05 was considered significant. No adjustment was made for multiple comparisons, as the risk of a type II error was felt to outweigh that of a type I error in this exploratory analysis. Results. Patients with platelet dysfunction were more severely injured (median Injury Severity Score 29.5 vs. 13.5, p=0.002) but otherwise demographically similar to those with retained platelet function. Of 232 proteins identified, twelve were significantly different in the low- vs. high-platelet function groups: gelsolin (median peak intensity 9.70E+6 vs. 1.48 E+7, p=0.002), transketolase (2.86E+4 vs. 9.36E+3, p=0.003), protein S100-A8 (4.60E+4 vs. 2.64E+4, p=0.006) and -A9 (5.83E+4 vs. 4.07E+4, p=0.012), histone H4 (5.72E+4 vs. 8.47E+6, p=0.007), histidine-rich glycoprotein (HRG) (5.00E+6 vs. 8.30E+6, p=0.008), factor XIII B chain (1.09E+6 vs. 1.61E+6, p=0.020), apolipoprotein A-IV (2.30E+7 vs. 3.28E+7, p=0.024), alpha-enolase (1.02E+5 vs. 4.15E+4, p=0.024), heat shock protein 90-alpha (1.41E+4 vs. 4.08E+3, p=0.045), alpha-2-HS-glycoprotein (2.83E+7 vs. 3.68E+7, p=0.045), and neutrophil gelatinase-associated lipocalin (3.44E+4 vs. 1.36E+4, p=0.045). These results are summarized in the figure, which shows one group of proteins that decreased in abundance (on the left side of the volcano plot) and another that increased (on the right side) in the patients with low platelet function. The twelve proteins that changed the most fall into several categories related to platelet function. Low gelsolin and high histone levels are each associated with microvascular obstruction by release of intracellular material that accumulates in small vessels and activates platelets. Low HRG and high damage-associated molecular pattern (DAMP) protein levels are consistent with massive innate immune activation, which can impair platelet function in many ways. Conclusion. This study provides an unbiased description of the change in proteomic profile associated with platelet dysfunction after trauma and identifies twelve proteins with the most profound changes. The pathways involving these proteins are salient targets for immediate investigation to better understand platelet dysfunction after trauma and identify targets for intervention. Figure Legend. Volcano plot depicting fold-change and statistical significance of individual proteins in patients with low vs. high platelet function. Dotted horizontal line represents p = 0.05. Twelve proteins showed a statistically significant p value. Figure Disclosures No relevant conflicts of interest to declare.

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M6PR-Specific Targeting of Lysosomal Heparanase Regulates Platelet Hemostatic Function in Mice

Blood ◽

10.1182/blood-2019-129075 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1059-1059

Author(s):

Nathan Eaton ◽

Caleb Drew ◽

Theresa A. Dlugi ◽

Karin M. Hoffmeister ◽

Hervé Falet

Keyword(s):

Platelet Function ◽

Conflicts Of Interest ◽

Blood Platelets ◽

Critical Role ◽

Fluorescent Microscopy ◽

Type I ◽

Dense Granules ◽

Specific Targeting

Besides α-granules and dense granules, which play critical roles in and beyond hemostasis, circulating blood platelets and their precursor cells megakaryocytes contain lysosomes, the contents of which are also secreted during platelet activation. In their delivery to the lysosome, acid hydrolases bearing phosphomannosyl residues are trafficked from the trans-Golgi network to the acidic late-endosomal compartment via the mannose 6-phosphate receptor (M6PR) pathway. To determine the role of M6PR-specific targeting of lysosomal enzymes in platelet function, platelet parameters were investigated in M6pr-/- mice lacking the 46-kDa M6PR, the physiological role of which is unclear. M6pr-/- mice had normal platelet count but displayed an increased number of distinct proplatelet-like cells compared to control mice, as determined by immunofluorescent microscopy. Moreover, transmission electron microscopy revealed the presence of abnormal membrane tubulations, elongated and electron-dense granules, and large vacuole-like structures within resting M6pr-/- platelets. M6pr-/- platelets expressed normally major glycoproteins on their surface and von Willebrand factor and fibrinogen in their α-granules. M6pr-/- mice were hyper-thrombotic, as assessed by tail bleeding time, and M6pr-/- platelets adhered to type I collagen with a significantly greater propensity than control platelets under arterial shear in in vitro flow experiments. Heparanase, an endo-β-glucuronidase that cleaves extracellular matrix heparan sulfate proteoglycans, is the most abundant lysosomal enzyme in platelets. Thus, its contribution to the phenotype of M6pr-/- mice was investigated further. Heparanase expression was decreased in the bone marrow megakaryocytes and blood platelets of M6pr-/- mice and increased in M6pr-/- plasma, as evidenced by immunoblot and fluorescent microscopy analysis, consistent with its mistargeting in the absence of M6PR. Interestingly, pharmacological inhibition of heparanase with OGT 2115 normalized the adhesion of M6pr-/- platelets to collagen in vitro, indicating that increased plasma heparanase contributes to the thrombotic phenotype of M6pr-/- mice. Taken together, the data suggest that the M6PR-specific targeting of lysosomal heparanase plays a critical role in platelet function, thereby regulating hemostasis. Disclosures No relevant conflicts of interest to declare.

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Quality Assurance At the Point of Care: PT/INR: What Is the Gold Standard for Patient Treatment?.

Blood ◽

10.1182/blood.v120.21.3170.3170 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3170-3170

Author(s):

Michael Plietz ◽

Aaron Leifer ◽

Amy S Fox ◽

Khrishan Naraine ◽

Vilma Padilla ◽

...

Keyword(s):

Therapeutic Range ◽

Gold Standard ◽

Conflicts Of Interest ◽

Point Of Care ◽

Patient Treatment ◽

P Value ◽

Optimal Conditions ◽

Sample Collection ◽

Blood Draw ◽

Coaguchek Xs

Abstract Abstract 3170 Background: The Prothrombin time (PT) can be performed in laboratories by instruments such as BCS (Siemens), and at the Point of Care using devices such as the Protime 3 (ITC) and the Coaguchek XS (Roche). Laboratory based instruments such as the BCS have been regarded as the Gold Standard for PT/INR assuming results can be obtained in under 24 hours from blood drawn in an uncentrifuged sample. A previous study performed in the Netherlands recommends that INR testing be completed within 6 hours of blood draw. This recommendation has not been universally adopted. In order to assess this effect, a study comparing the laboratory instrument (BCS) to POC instruments was performed while simultaneously comparing lab based results at or greater than 6 hours. Methods: The INR values of the BCS in two separate laboratories, Coaguchek XS, and Protime 3 were compared to each other. The INR values and turnaround times for each were recorded. The results were compared using paired observation T-tests and regression analysis including all data and within sub-therapeutic (<2), therapeutic (2.0–3.0), and supratherapeutic range (>3.0). Additionally, point of care results were compared to BCS test results before and after 6 hours. Results: 60 patients had 3 INR test methods performed. All samples were tested in two different reference laboratories, both using BCS testing method. Reference lab A performed the tests under optimal conditions including a turnaround times of less than 2 hours, while Reference lab B performed the tests under usual workflow conditions. While the total Protime 3 results were significantly different from Reference lab A, the Coaguchek XS results were not. In the therapeutic range, the Coaguchek XS and Reference lab B had significant correlation (r = .556 and .634 respectively) to reference lab A while the Protime 3 did not (r = .281). Reference lab B results were significantly different from Reference lab A when testing was performed after 6 hours (P-value = .0252), but had similar results to Reference lab A when performed within 6 hours (P-value = .402). The Coaguchek XS showed no significant differences when compared to Reference lab A results (p-value = .1262). Another indicator of lack of concordance between Reference lab A and B, when testing is performed greater than 6 hours, was the change in directionality of the bias (p-value = .0396). Additionally, Protime 3 results in the therapeutic range correlated significantly better with Reference lab B results when they were resulted within 6 hours (r= .191 and r= .782). Conclusions: Based on these findings, the BCS method appears to lose reliability when the test is performed on uncentrifuged blood greater than 6 hours after blood draw. Interestingly, the Coaguchek XS correlated best with Reference lab A, BCS testing under optimal conditions. The Protime 3, on the other hand, was less well correlated with Reference lab A. The change in directionality of the bias also suggests that the BCS results were flawed when testing was performed after 6 hours. Although additional preanalytic factors (i.e. temperature) may have had an effect on these results, these data suggest that laboratory based INR testing should be performed within six hours of sample collection. Disclosures: No relevant conflicts of interest to declare.

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In Vitro Characterization of Platelet Dysfunction In Common Hematological Disorders Using the Verify Now® Assay

Blood ◽

10.1182/blood.v116.21.1436.1436 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1436-1436

Author(s):

Nina D’Abreo ◽

Vatsala Kirtani ◽

Jennifer L. Kujawa Eisenstein ◽

Yelda Nouri ◽

Matt Geller ◽

...

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Reference Ranges ◽

Platelet Dysfunction ◽

Platelet Defects ◽

Verify Now ◽

Instructions For Use

Abstract Abstract 1436 Objectives: Myelodysplastic syndrome (MDS), immune thrombocytopenia purpura (ITP) and myeloproliferative disorders (MPD) are frequent diagnoses in a community hematology practice. Qualitative platelet defects are common features of these disorders but are difficult to characterize in daily practice as specialized technology is not readily available. The Verify Now® (VN) is a rapid, point-of service assay to measure platelet aggregation. VN assesses the degree of inhibition of GPIIb/IIIa mediated aggregation and uses minimal sample preparation. Its main application currently, is to determine the adequacy of anti-platelet therapy with aspirin (ASA), thienopyridines, such as clopidogrel (CPL), and GPIIb/IIIa inhibitors in patients with cardiovascular disease. (Verify Now for IIb/IIIa, aspirin, and P2Y12, Instructions for Use, Accumetrics) We used VN to detect platelet aggregation defects in patients with ITP, MDS and MPD and compared the to a platelet function analyzer PFA -100. (Francis JL; In Michelson AD, ed. Platelets, 2nded. San Diego: Elsevier/Academic Press, 2007:535-544) Methods: Subjects with MDS, ITP, and MPD were identified from our Hematology practice. Informed consent was obtained from all patients. Patients taking aspirin and /or clopidogrel were not excluded. Platelet function was determined concurrently using VN and PFA-100 using standard reference ranges. (Verify Now for IIb/IIIa, aspirin, and P2Y12, Instructions for Use, Accumetrics), (Mammen EF et al; Semin Throm Hemost. 1998; 24 (2):195-205). Results: Thirty three patients are enrolled in the study, 21 with ITP, 6 with MDS and 6 with MPD. Assay results are available for 30 patients. Platelet counts for all patients ranged from 2 to 1206 ×103/cmm, mean platelet count being 184 ×103/cmm (SD ± 215) and median count was 120 ×103/cmm. ITP: In 15 patients with ITP and platelet count ≥ 100 ×103/cmm, 11 were not on ASA/CPL. PFA-100 and VN each detected abnormalities in 4 patients but results were non-concordant in 2 patients. In 4 patients on ASA, VN detected ASA effect in all 4 whereas the PFA-100 detected 2. In 2 patients with platelet count < 100 ×103/cmm not on ASA/CPL, VN detected abnormalities in both, whereas PFA detected only 1. (Table 1) MDS: Two patients had counts ≥ 100 ×103/cmm. One was on ASA and both assays detected ASA effect. Neither assay detected an abnormality in the patient not on ASA. In 4 patients with counts <100 ×103/cmm, 3 were not on ASA/ CPL. Both assays detected abnormalities in all 3 and ASA effect in 1 patient on ASA. (Table 2) MPD: In 4 patients with counts < 400 ×103/cmm, not on ASA/CPL, both assays detected non-concordant abnormalities in 1 patient. Both assays detected CPL and ASA effect in 1 patient taking both drugs. 2 patients had count ≥400,000. In 1 patient taking CPL, VN detected CPL effect but PFA did not. In 1 patient on ASA, PFA detected abnormality but VN did not. (Table 3) Conclusion: In patients with MDS, ITP, and MPD, abnormal platelet function is common. These results show that VN may be as sensitive for detecting platelet dysfunction as PFA-100. Results were especially concordant in MDS patients. We plan to include 150 patients to confirm these results. We hope to define qualitative platelet defects in this population so that appropriate intervention can be recommended prior to invasive procedures. Disclosures: No relevant conflicts of interest to declare.

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Dasatinib-Induced Platelet Dysfunction.

Blood ◽

10.1182/blood.v110.11.2941.2941 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2941-2941 ◽

Cited By ~ 3

Author(s):

Alfonso Quintas-Cardama ◽

Xin Han ◽

Hagop Kantarjian ◽

Jorge Cortes

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Function Analysis ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Light Transmittance ◽

P Value ◽

Platelet Dysfunction

Abstract Dasatinib is efficacious and safe in patients (pts) with chronic myelogenous leukemia (CML) after imatinib failure. However, dasatinib therapy has been linked to bleeding in some pts, typically affecting mucosal surfaces. We analyzed the effect of dasatinib on basic coagulation tests and platelet aggregation function in 55 pts with CML in chronic phase at diagnosis (1), >8 weeks after tyrosine kinase inhibitor (TKI) discontinuation (3), or during therapy with the ABL/SRC inhibitors dasatinib (n=27) and bosutinib (n=12), or the aminopyrimidine derivatives imatinib (n=8) and nilotinib (n=4). Median age was 54 yrs (range, 22–80), WBC 5.5 ×109/L (range, 2.1–58.5), hemoglobin 12.2 g/dL (range, 9.7–15.1), and platelets 230 ×109/L (range, 80–746). Only 2 pts (both on imatinib and warfarin) had abnormal basic coagulation parameters (prolonged prothrombin time [PT]). In the remainder 53 pts, the median PT time was 11.6 sec (9.8–13), activated partial thromboplastin time (aPTT) 28.5 sec (range, 21–33.9), and fibrinogen level 495 mg/dL (range, 336–700), suggesting an intact secondary hemostasis. To evaluate the primary hemostasis status, platelet aggregation was measured by turbidometry in 1 mL of platelet-rich plasma upon stimulation with 5 μM of ADP, 1 μg/ml of collagen, 1 μM of epinephrine, 1 mM arachidonic acid, or 8 μL of ristocetin by using a Whole Blood Lumi-Aggregometer (Chrono-Log Corporation, Havertown, PA). Aggregation was expressed as the maximal percent change in light transmittance from baseline using platelet-poor plasma as a reference. Five (9%) patients (2 bosutinib, 1 nilotinib, and 2 imatinib) were on aspirin and showed an aspirin-like effect (decreased platelet aggregation with arachidonic acid [AA] and epinephrine [Epi]). Patients off-TKI or untreated had a normal platelet aggregation test. Results on the remaining 46 assessable patients demonstrate that dasatinib is strongly associated platelet aggregation defects, particularly an aspirin-like effect, when compared with other TKIs. Platelet function analysis by PFA-100 showed that addition of dasatinib (400 nM) to fresh whole blood obtained from a healthy volunteer rapidly induced platelet dysfunction. Closure times were prolonged from 100 sec (baseline) to >300 sec in response to Epi (normal <160 sec) but not in response to ADP (from 72 to 84 sec, normal <120 sec) after 30 min incubation. In summary, our data indicate that dasatinib does not interfere with secondary hemostasis. Rather, it inhibits platelet function in an aspirin-like manner. This effect, while independent, can be additive to dasatinib-induced thrombocytopenia and may account for the bleeding diathesis observed in pts with CML receiving dasatinib. Table 1. Effect of BCR-ABL kinase inhibitors on platelet aggregation Dasatinib Bosutinib Nilotinib or Imatinib Platelet aggregation test n=27 n=10 n=9 p value Normal (%) 4 (15) 8 (80) 6 (67) < 0.01 Abnormal (%) 23 (85) 2 (20) 3 (33) < 0.006 Only with Epi 7 1 0 With both Epi and AA 14 1 3 With all agonists 2 0 0

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Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway

Blood ◽

10.1182/blood-2021-152047 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3246-3246

Author(s):

Filip Radenkovic ◽

Sara Chiaretti ◽

Mark Burton ◽

Penny Hassell ◽

Xuan Bui ◽

...

Keyword(s):

Flow Cytometry ◽

Conflicts Of Interest ◽

Single Amino Acid ◽

Blue Staining ◽

Human Neutrophils ◽

P Value ◽

Type I ◽

Type Ii ◽

Extracellular Loop

Abstract INTRODUCTION: The human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model. STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test. RESULTS AND DISCUSSION: GAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001). CONCLUSIONS: In this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Genetic Variants In Platelet Supervillin but Not Archvillin Are Associated with PFA-100 Closure Times In African Americans

Blood ◽

10.1182/blood.v116.21.4311.4311 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4311-4311

Author(s):

Ying Jin ◽

Daniel Covarrubias ◽

Altaf A Kondkar ◽

Srikanth Nagalla ◽

Elizabeth J Luna ◽

...

Keyword(s):

Myocardial Infarction ◽

Shear Stress ◽

African Americans ◽

Platelet Function ◽

Genetic Variants ◽

Conflicts Of Interest ◽

Focal Adhesions ◽

Cell Spreading ◽

P Value ◽

Mrna Levels

Abstract Abstract 4311 Myocardial infarction typically occurs after atherosclerotic plaque rupture that exposes collagen under high shear stress, favoring platelet adhesion, activation with ADP release, aggregation and platelet thrombus formation. There is a well-established genetic contribution to myocardial infarction, and we have previously shown a shear-dependent assay of platelet function is heritable (JTH 2007;5:1617). Collagen-induced platelet aggregation is highly heritable in African Americans (AAs) but not in European Americans, which may be relevant since AAs have the highest overall coronary heart disease mortality rate of all ethnic groups in the US. The mechanisms for these ethnic differences in platelet function are not clear. The goal of the current study was to identify genetic variants in AAs affecting the Platelet Function Analyzer (PFA)-100 (ADP cartridge) that is both collagen- and shear-dependent. We recruited healthy AA subjects and measured closure times in the PFA-100. Subjects were excluded if there was evidence of exposure to anti-platelet therapy (absent response to arachidonic acid). Closure times showed a normal distribution (mean CT = 96 sec +/− 20 sec [SD]). Genome-wide genotyping with the Infinium II Assay using the Illumina BeadStation 500 system was performed. After controlling for population admixture and excluding markers with a less than 95% call rate, 131 subjects passed criteria for ethnicity and successful genotyping of 956,604 tagSNPs. Statistical analysis identified a locus on chromosome 10 that appeared to be associated with PFA-100 closure times. Of the 7 most significantly associated SNPs, 5 were in SVIL, the gene encoding supervillin. The closest flanking genes to the SNP with the best P value were >77 kb upstream (2 pseudogenes) and >214 kb downstream (LTZ1; lysozyme-like 1). After adjusting for VWF and fibrinogen levels, rs10826650 in SVIL was associated with PFA-100 closure times (P = 5.92E–07). We pursued SVIL (chr 10p11.23) because it is known to form a high-affinity link between the actin cytoskeleton and the plasma membrane during cell spreading and disassembly of focal adhesions. Supervillin slows the rate of cell spreading and increases myosin contractility by linking the myosin II heavy chain with myosin light chain kinase. Supervillin also decreases the stability of focal adhesions and co-localizes with signaling molecules in cholesterol-rich lipid rafts. Prior work has identified 2 transcript variants encoding different isoforms of supervillin. Isoform 1 (supervillin) is reported to be most abundantly expressed in muscle, bone marrow, thyroid gland and salivary gland; isoform 2 (archvillin) is reported as muscle specific. Because neither isoform has been characterized in platelets, we performed gene expression analysis with 23 leukocyte depleted platelet (LDP) RNA samples (<1 leukocyte per 5 million platelets) using the Sentrix BeadChip and BeadStation system from Illumina, Inc. A probe recognizing both isoforms was highly expressed in these samples, whereas a probe specific for the archvillin isoform was barely detectable. The level of SVIL mRNA expression was higher than PECAM1 but less than GPIbα. These microarray data were confirmed by RT-PCR using LDP RNA and PCR primers specific for supervillin. Notably, SVIL was not detected in megakaryocyte lines, HEL or Meg-01. Anti-supervillin antisera recognized the appropriate sized ∼205 kD polypeptide by immunoblotting. PFA-100 closure times were found to directly correlate with supervillin (r = 0.481, P = 0.02) but not with archvillin (r = 0.11, P=n.s.) mRNA levels. In summary, we have demonstrated for the first time that platelets contain supervillin but not the alternately spliced isoform archvillin, and that expression is associated with PFA-100 closure times in AAs. Because higher supervillin expression was associated with longer closure times, this finding is consistent with an inhibitory role for supervillin in regulating the rapid actin cytoskeletal rearrangement required to form platelet thrombi on collagen under shear stress. Disclosures: No relevant conflicts of interest to declare.

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Platelet activation and reactivity in a large cohort of patients with Gaucher disease

Thrombosis and Haemostasis ◽

10.1055/a-1642-4206 ◽

2021 ◽

Author(s):

Shoshana Revel-Vilk ◽

Mira Naamad ◽

Dafna Frydman ◽

Michael R. Freund ◽

Tama Dinur ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Function ◽

Gaucher Disease ◽

P Value ◽

Bleeding Tendency ◽

Platelet Dysfunction ◽

Platelet Surface ◽

Increased Risk ◽

Αiibβ3 Integrin ◽

Cd63 Expression

Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data. Methods: Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. Results: GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels (r 0.17, p-value 0.042). Splenectomized GD patients had a higher level of unstimulated αIIbβ3 integrin and P-selectin expression. Reduced platelet reactivity (-2 SD of reference range) was found in 79 (53%, 95% CI 44%-61%) patients, of whom 10 (6.7%, 95% CI 3.3%-12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (OR (95% CI), 1.05 (1.01-1.1)) and low P-selectin reactivity (OR (95% CI), 2.03 (1.25-3.35)) were associated with more than one bleeding manifestation. Conclusion: Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not αIIbβ3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

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Evaluation of the Abnormal Platelet Function in von Willebrand Disease by the Blood Filtration Test

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650600 ◽

1996 ◽

Vol 76 (03) ◽

pp. 460-468 ◽

Cited By ~ 7

Author(s):

Francesco I Pareti ◽

Marco Cattaneo ◽

Luca Carpinelli ◽

Maddalena L Zighetti ◽

Caterina Bressi ◽

...

Keyword(s):

Platelet Function ◽

Relevant Information ◽

Von Willebrand Disease ◽

Type I ◽

Cumulative Number ◽

Primary Hemostasis ◽

Normal Platelet ◽

Filter Test ◽

Type 3 ◽

Von Willebrand

SummaryWe have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillarysized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with l-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VUI/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ibα and Ilb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.

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Bleeding Time in Rats: A Comparison of Different Experimental Conditions

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657230 ◽

1982 ◽

Vol 48 (01) ◽

pp. 108-111 ◽

Cited By ~ 118

Author(s):

Elisabetta Dejana ◽

Silvia Villa ◽

Giovanni de Gaetano

Keyword(s):

Platelet Function ◽

Bleeding Time ◽

Platelet Dysfunction ◽

Experimental Conditions ◽

Platelet Number ◽

Coagulation Defects ◽

Tail Tip ◽

And Function ◽

Storage Pool Disease ◽

Type Of Anaesthesia

SummaryThe tail bleeding time (BT) in rats definitely varies according to the method applied. Of the various variables that may influence BT, we have evaluated the position (horizontal or vertical) of the tail, the environment (air or saline), the temperature (4°, 23° or 37° C) and the type of anaesthesia. Transection of the tail tip cannot be used to screen drugs active on platelet function since it is sensitive to coagulation defects. Template BT in contrast is not modified by heparin and is sensitive to defects of platelet number and function (“storage pool disease”, dipyridamole-like drugs, exogenous prostacyclin). In contrast the test fails to detect aspirin-induced platelet dysfunction. The evidence reported indicates that thromboxane A2-prostacyclin balance is not a factor regulating BT. Aspirin treatment however may be a precipitating factor when associated with other abnormalities of platelet function.Template BT is a valid screening test for platelet disorders and for antiplatelet drugs.

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Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-01912-3 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Shunqiao Feng ◽

Lin Han ◽

Mei Yue ◽

Dixiao Zhong ◽

Jing Cao ◽

...

Keyword(s):

Next Generation Sequencing ◽

Langerhans Cell Histiocytosis ◽

Mutation Screening ◽

Langerhans Cell ◽

Braf V600e ◽

P Value ◽

Type I ◽

Next Generation ◽

Mutation Status ◽

Generation Sequencing

Abstract Background Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

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