Influence of Nanopolymers with Different End-Functionalities on Platelet Function and the Coagulation Cascade - An Ex-Vivo Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4038-4038
Author(s):  
Meera Chitlur ◽  
Erin Ware ◽  
Sujata Kannan ◽  
Wendy Hollon ◽  
Steve Buck ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Surface Charge ◽  
Platelet Function ◽  
Whole Blood ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Cancer Drug ◽  
Dendritic Polymers ◽  
Drug Concentrations

Abstract Dendritic polymers are branched nanopolymers with a central core and multiple peripheral functional groups that offer great potential as high payload delivery vehicles carrying multiple copies of drug molecules, targeting ligands and imaging agents to their site of action. Their nanoscopic dimensions offer exciting possibilities for achieving high intracellular drug concentrations in many therapeutic areas including anti-cancer drug delivery. Biocompatibility and biodistribution of dendritic polymers may be influenced by surface charge and concentration. One of the major challenges in their use is the effect on coagulation. The objective of this study was to determine the effect of change in surface charge and concentration of dendritic polymer on cellular and enzymatic components of coagulation. Materials and Methods: The effect of increasing concentrations (1, 10, 100, and 1000mcg/ml) of polyamidoamine (PAMAM) dendrimers with -COOH (anionic), -OH (neutral), and -NH2 (cationic) end functionalities, on platelet function and coagulation was evaluated using thromboelastography, whole blood aggregation, and flow cytometry. The thromboelastographic profile and platelet aggregation studies were obtained on samples of whole blood incubated for thirty minutes with dendrimer. Platelets were incubated with FITC labelled dendrimer for 30,60 and 120 mins, to determine uptake and platelet activation using flow cytometry. All tests were performed in triplicate. RESULTS: Thromboelastography: No significant effect on clot formation (time to clot formation and size) was seen with PAMAM-COOH (COOH) or PAMAM-OH (OH). Prolonged time to initiation of clot and decreased size were noted with 100 and 1000mcg/ml of PAMAM-NH2(NH2) as shown in figure1, indicating impairment of both the enzymatic and cellular components of the coagulation system. Whole Blood Aggregation: Neither platelet aggregation nor secretion were significantly affected by COOH or OH. Platelet aggregation was significantly decreased with NH2 at 100 and 1000mcg/ml. Flow Cytometry: Spontaneous CD62 activation was seen in platelets incubated with NH2. No spontaneous CD62 activation was noted with COOH or OH even at 1000mcg/ml. Platelet uptake of FITC labeled dendrimer was assessed at 30, 60 and 120mins of incubation. Increased uptake of FITC labeled dendrimer was noted at 2 hours with NH2. TEG clotting Profiles with PAMAM-NH2. TEG clotting Profiles with PAMAM-NH2. CONCLUSIONS: Surface charge of the dendritic nanopolymers plays a significant role on its effect on coagulation and platelet function. The anionic -COOH terminated and neutral -OH terminated dendrimers had no effect on hemostasis even at the highest concentrations while the cationic-NH2 was associated with inhibition of platelet aggregation and delayed clot initiation at higher concentrations. This would indicate that the anionic and neutral dendrimers would serve as better vehicles than cationic dendrimers for targeted delivery of therapeutic agents.

scholarly journals Analysis of blood clotting with the total thrombus analysis system in healthy dogs

2021 ◽  
pp. 104063872199186
Author(s):  
Tomoko Iwanaga ◽  
Ryuji Fukushima ◽  
Tomoka Nagasato ◽  
Ikuro Maruyama ◽  
Naoki Miura
Keyword(s):  
Blood Flow ◽  
Platelet Function ◽  
Whole Blood ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Test Pressure ◽  
Analysis System ◽  
The Right

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.

Effects of Angiotensin-Converting Enzyme Inhibition on Thromboelastogaphy for Infants and Children Presenting for Elective Cardiac Surgery.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1225-1225
Author(s):  
Michael L Schmitz ◽  
Edgar D St. Amour ◽  
Kimo Stine ◽  
Mark A Austen ◽  
Chanhee Jo ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Whole Blood ◽  
Infants And Children ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Angiotensin Converting Enzyme Inhibition ◽  
Clot Strength ◽  
Elective Cardiac Surgery

Abstract Introduction: Thromboelastography (TEG) is a point-of-care whole blood coagulation test used commonly with cardiac and liver operations, surgeries for which intraoperative and postoperative bleeding and coagulopathies occur and transfusion is likely. Angiotensinconverting enzyme inhibitors (ACEI) have been found to decrease plasminogen activator inhibitor I (PAI-1), increase tissue plasminogen activator (tPA), decrease tissue factor (TF) production by monocytes, and decrease platelet aggregation. Methods: With IRB approval, kaolin-activated heparinase TEG data were collected on 634 infants and children under 18 yr presenting for elective cardiac surgery between January 2004 and December 2007 for retrospective review utilizing the Haemoscope Thromboelastograph® Coagulation Analyzer (Haemoscope Corp., Niles, IL). Six TEG parameters are analyzed: R (reaction time to reach clot initiation), K (time to reach 20 mm amplitude after R), alpha (clot kinetics), MA (maximum amplitude), LY30 (amount of lysis 30 min after MA), and CI (coagulation index). Infants and children are analyzed based on preoperative therapy with an ACEI. Results: In those receiving ACEI, the time to initial fibrin formation is faster (R, p=0.0007), although subsequent clot formation is slower (K, p=0.0201; alpha, p=0.0092) than without ACEI drugs. Moreover, there is markedly less fibrinolysis (LY30, p=0.0019) in those receiving an ACEI. Clot strength and overall clot dynamics (MA and CI, p=NS) were similar in both groups (Table 1). Subjects with baseline oxygen saturation of 88% differ in that they do not have a significant change in K or alpha but do have a higher CI. Table 1. Ranges for Kaolin-activated Heparinase Thromboelastography Parameters Group R (min) K (min) alpha (degrees) MA LY30% CI Control 7.27±2.20 1.83±0.68 65.0±8.4 64.2±6.4 2.28±2.87 −0.68±2.81 ACEI 6.54±1.88 1.99±0.81 62.7±10.2 63.3±5.8 1.12±2.05 −0.39±2.87 p value 0.0007 0.0201 0.0092 NS 0.0019 NS Discussion: A number of investigators have found antithrombotic effects of ACEI drugs in adults but children have not been studied. The TEG provides an ex vivo means of assessing whole blood clotting dynamics. This preliminary analysis of infants and children reveals a delay in the time required to initiate clot via the intrinsic coagulation cascade as activated by kaolin (R) and slowed clot formation (K and alpha). Final clot strength shows no difference and LY30 shows less fibrinolysis. A shift in plasma hemostasis towards fibrinolysis would potentially delay R, prolong K, decrease alpha, and increase LY30. Paradoxically, we see a decrease in R and LY30. Reduced platelet aggregation is consistent with slowed kinetics of clot formation (K and alpha) but would be expected to cause a reduction in MA that is not seen. Further analysis of this data will separate the subjects into age groups as TEG parameters vary with age in infants and children. Also, since there is evidence that enalapril may not have the antiplatelet effects of captopril and may increase tPA in women but not men, further analysis of gender and type of ACEI given will be completed.

Properties of Fibrinogen Cleaved by Jararhagin, a Metalloproteinase from the Venom of Bothrops jararaca

1994 ◽  
Vol 72 (02) ◽  
pp. 244-249 ◽  
Author(s):  
Aura S Kamiguti ◽  
Joseph R Slupsky ◽  
Mirko Zuzel ◽  
Charles R M Hay
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Human Fibrinogen ◽  
Activated Platelets ◽  
Bothrops Jararaca ◽  
Platelet Binding ◽  
Fibrin Monomers ◽  
Fibrinogen Molecule

SummaryHaemorrhagic metalloproteinases from Bothrops jararaca and other venoms degrade vessel-wall and plasma proteins involved in platelet plug and fibrin clot formation. These enzymes also cause proteolytic digestion of fibrinogen which has been suggested to cause defective platelet function. Fibrinogen degradation by jararhagin, a metalloproteinase from B. jararaca, and the effect of jararhagin fibrinogenolysis on both platelet aggregation and fibrin clot formation were investigated. Jararhagin was found to cleave human fibrinogen in the C-terminal region of the Aα-chain giving rise to a 285-290 kDa fibrinogen molecule lacking the Aα-chain RGD 572-574 platelet-binding site. Platelet binding and aggregation of ADP-activated platelets is unaffected by this modification. This indicates that the lost site is not essential for platelet aggregation, and that the remaining platelet binding sites located in the N-terminal portion of Aα chains (RGD 95-97) and the C-terminal of γ chains (dodecapeptide 400-411) are unaffected by jararhagin-digestion of fibrinogen. Fibrin clot formation with thrombin of this remnant fibrinogen molecule was defective, with poor polymerization of fibrin monomers but normal release of FPA. The abnormal polymerization could be explained by the loss of one of the two complementary polymerization sites required for side-by-side association of fibrin protofibrils. Jararhagin-induced inhibition of platelet function, an important cause of haemorrhage in envenomed patients, is not caused by proteolysis of fibrinogen, as had been thought, and the mechanism remains to be elucidated.

The Haemocompatibility of Biomaterials In Vitro: Investigations on the Mechanism of the Whole-blood Clot Formation Test

1992 ◽  
Vol 20 (3) ◽  
pp. 390-395 ◽  
Author(s):  
Thomas Groth ◽  
Katrin Derdau ◽  
Frank Strietzel ◽  
Frank Foerster ◽  
Hartmut Wolf
Keyword(s):  
Whole Blood ◽  
Blood Clotting ◽  
Blood Clot ◽  
Formation Rate ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Contact Activation ◽  
Human Fibrinogen ◽  
Static Conditions

Twenty years ago Imai & Nose introduced a whole-blood clotting test for the estimation of haemocompatibility of biomaterials in vitro In our paper a modification of this assay is described and the mechanism of clot formation further elucidated. It was found that neither the inhibition of platelet function nor the removal of platelets from blood significantly changed the clot formation rate on glass and polyvinyl chloride in comparison to the rate tor whole blood. Scanning electron microscopy demonstrated that platelets were not involved in clot formation near the blood/biomaterial interface. Thus, it was concluded that the system of contact activation of the coagulation cascade dominates during clot formation under static conditions. The latter conclusion was supported by the fact that preadsorption of human serum albumin or human fibrinogen onto the glass plates used, decreased the clot formation rate in the same manner.

Enhanced platelet aggregation and activation under conditions of hypothermia

2007 ◽  
Vol 98 (12) ◽  
pp. 1266-1275 ◽  
Author(s):  
Ruben Xavier ◽  
Ann White ◽  
Susan Fox ◽  
Robert Wilcox ◽  
Stan Heptinstall
Keyword(s):  
Cardiac Surgery ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Baseline Levels ◽  
Spontaneous Aggregation ◽  
High Concentrations ◽  
Induced Aggregation ◽  
P2y12 Antagonist

SummaryThe effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28°C and 37°C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28°C compared with 37°C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28°C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28°C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28°C compared with 37°C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28°C, but reversal of aggregation was seen at 37°C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28°C compared with 37°C. Expression was significantly increased at 28°C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28°C compared with 37°C, particularly in the presence of ADP.

Rapid Assessment of Platelet Function Using Thromboelastography and Small Volumes of Citrated Whole Blood.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3995-3995 ◽  
Author(s):  
Fred G. Pluthero ◽  
Margaret L. Rand ◽  
Victor S. Blanchette ◽  
Walter H. Kahr
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Function Analysis ◽  
Maximum Amplitude ◽  
Ease Of Use ◽  
Platelet Response ◽  
Platelet Function Disorders ◽  
Wide Range

Abstract Platelet function disorders are a key cause of abnormal bleeding, and diagnosis is challenging because: platelet abnormalities are diverse, affecting many aspects of function; variability in platelet function testing in clinical laboratories makes it difficult to compare results; large blood volumes required for platelet function analysis make it difficult to perform in neonatal patients; manipulation of platelet rich plasma used for platelet aggregation can lead to test variability; platelet aggregation curves are difficult to interpret in thrombocytopenic patients. We describe a method of testing platelet function using citrated whole blood and thromboelastography (TEG) that overcomes some of these limitations. Commercially-available platelet mapping kits allow the effects of the platelet agonists adenosine diphosphate (ADP) and arachidonic acid (AA) to be assessed via a TEG assay where reptilase and activated factor XIII produce fibrin clots independent of thrombin in heparinized whole blood. The activation and aggregation of platelets is quantified by measuring the difference in maximum amplitude (MA) between unstimulated samples, which form weak fibrin-only clots, and samples with agonists added, which form stronger clots containing fibrin and activated/aggregated platelets. Platelet mapping was used as the basis for a TEG assay which can be used to assess platelet responses to a wide range of stimuli - including ADP, AA, epinephrine, collagen, U46619 (thromboxane-A2 receptor agonist), SFLLRN (PAR-1 thrombin receptor activating peptide) and AYPGKF (PAR-4 activating peptide) - in small samples (330μL) of citrated native (CN) blood or plasma to which heparin is added to a concentration of 20U/mL. Samples were recalcified by adding calcium chloride to 10mM (necessary for the function of reptilase and FXIIIa), and other reagent volumes were the same as in platelet mapping assays, with fibrin activator prepared at 1/2 regular strength. The concentrations of platelet agonists were: collagen 51μg/ml, epinephrine 0.27μM, ADP 5.4μM, arachidonic acid 135μg/mL, U46619 2.6μM, SFLLRN 6.76μM and AYPGKF 34μM. These concentrations produced TEG MA values in heparinated fibrin-activated CN blood from a panel of normal individuals comparable to those obtained from recalcified CN blood in the absence of heparin (the fibrin/platelet response control). The platelet response was rapid with maximum amplitudes reached within 10 minutes for all agonists except collagen, which required >30 minutes to produce maximum amplitude. We have found this TEG platelet-response assay to be useful in detecting platelet function abnormalities, producing results which correlate with and extend those of other platelet function tests. For example in one patient a weak response to epinephrine corresponded to similar platelet aggregation results, and in another the TEG assay detected a weak PAR-1 response not specifically detected in other tests. The assay has also proven useful in assessing platelet function in blood and plasma having low platelet concentrations (<50 x 10E9/L) from experimental or pathological causes (e.g. thrombocytopenia), in titrating platelet responses to agonists and in assessing the effects of antiplatelet agents in vivo and in vitro. Thus this TEG platelet function assay has the advantages of speed, ease of use, flexibility, adaptability to low platelet concentrations and sample economy, requiring small volumes of citrated blood which can be used for other coagulation assays and platelet response tests.

Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment

2008 ◽  
Vol 99 (01) ◽  
pp. 121-126 ◽  
Author(s):  
Siegmund Braun ◽  
Stefan Jawansky ◽  
Wolfgang Vogt ◽  
Julinda Mehilli ◽  
Albert Schömig ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Platelet Aggregometry ◽  
Light Transmission Aggregometry ◽  
Before And After ◽  
Standardized Method ◽  
Multiple Electrode

SummaryThe level of platelet aggregation, measured with light transmission aggregometry (LTA) in platelet rich plasma (PRP), has been shown to predict outcomes after percutaneous coronary intervention (PCI). However, measuring parameters of platelet function with LTA is time consuming and weakly standardized. Thus, a fast and standardized method to assess platelet function after clopidogrel treatment would be of great value for clinical practice. A new method, multiple electrode platelet aggregometry (MEA), to rapidly measure platelet aggregation in whole blood has recently been developed. The aim of this study was to assess parameters of platelet function with MEA and LTA before and after administration of 600 mg clopidogrel. Blood samples from 149 patients scheduled for coronary angiography were taken after clopidogrel treatment; in addition, in 60 of the patients samples were available before clopidogrel treatment. ADP-induced platelet aggregation was measured with LTA and simultaneously in whole blood with MEA on the Multiplate analyzer. Platelet aggregation measured with MEA decreased significantly after clopidogrel treatment (P<0.0001). ADP-induced platelet aggregation assessed with MEA and LTA correlated significantly (Spearman rank correlation coefficient=0.71; P<0.0001).The results of MEA, a fast and standardized method to assess the platelet response to ADP prior to and after clopidogrel treatment, correlate well with LTA.

scholarly journals Highly Procoagulant Extracellular Vesicles in Amniotic Fluid

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4961-4961
Author(s):  
Johannes Thaler ◽  
Lena Hell ◽  
Lukas Wisgrill ◽  
Andreas Spittler ◽  
Michael Schwameis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Amniotic Fluid ◽  
Blood Coagulation ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Blood Clotting ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Contact Activation ◽  
Plasma Factor

Abstract Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role. Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC. Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma. Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF. Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. Disclosures No relevant conflicts of interest to declare.

scholarly journals Flow Cytometry-Based Platelet Function Testing Is Predictive of Symptom Burden in a Cohort of Bleeders

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2255-2255
Author(s):  
Niklas Boknäs ◽  
Sofia Ramström ◽  
Lars Faxälv ◽  
Tomas L Lindahl
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Platelet Function ◽  
Whole Blood ◽  
Test Results ◽  
Platelet Function Testing ◽  
Fibrinogen Binding ◽  
Normal Test ◽  
Entire Patient Cohort ◽  
Function Testing

Abstract Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the clinical significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clear link between test results and patient phenotype. Herein, we present results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and p-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or the fifth percentile of the entire patient sample. We found that abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient sample (P = 0.00008, hazard ratio 8.7; 95 % CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing is a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related primary hemostasis. Figure 1. Test results for each patient in comparison with reference range (A) and the fifth percentile of the entire patient cohort (B). Normal test results are colored grey, abnormal test results are colored with a continuous color gradient using the deviation from the mean divided by the standard deviation as a measure of degree of abnormality. Grey horizontal bars illustrate the number of abnormal test results for each patient. Figure 1. Test results for each patient in comparison with reference range (A) and the fifth percentile of the entire patient cohort (B). Normal test results are colored grey, abnormal test results are colored with a continuous color gradient using the deviation from the mean divided by the standard deviation as a measure of degree of abnormality. Grey horizontal bars illustrate the number of abnormal test results for each patient. Disclosures Lindahl: Diapensia: Equity Ownership.

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