Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3391-3391
Author(s):  
Georgios Pongas ◽  
Swapan Dasgupta ◽  
Perumal Thiagarajan
Keyword(s):  
Conflicts Of Interest ◽  
Hospital Setting ◽  
Fluorescence Emission ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Fluorescence Emission Spectrum ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Gram Negative ◽  
Normal Controls

Abstract Abstract 3391 Introduction The anti-platelet factor 4(PF4)/heparin antibodies, arising as a result of previous heparin exposure, are causally related to the procoagulant state due to platelet and monocyte activation. Formation of these antibodies with subsequent thrombocytopenia or thrombosis has also been described in patients, who have not been previously exposed to heparin. The presence of anti-PF4/heparin antibodies in individuals correlates with the severity of periodontal disease, implying that their occurrence may be triggered by periodontal pathogens. In this study, we determined the presence of anti-PF4/heparin antibodies in gram-negative bacteremic patients in a hospital setting and propose a pathophysiologic mechanism of their presence. Method We developed an in house ELISA for quantifying anti-PF4/heparin antibodies using therapeutic heparin and PF4 isolated from platelets. We used serum from a patient with high optical density as a standard and assigned an unit of 100 arbitrarily to construct a standard curve. We tested the sera from gram negative bacteremic patients (n= 34) in the quantitative ELISA along with normal controls (n=10). We also developed an in house ELISA for studying cross reactivity between anti-PF4/heparin antibodies and lipopolysaccharide (LPS)/PF4. We tested the sera from patients (n=5) with heparin induced thrombocytopenia in this cross reactivity ELISA. To test the interaction of LPS with PF4, we labeled PF4 with Alexa488 and measured its binding to LPS by monitoring the changes in fluorescence emission spectrum following excitation at λ480. Results Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls (26.4 ± SD 33 units, N=34 versus 6.3 ± SD 2.38 units, N=10, P=0.032). Bacterial LPS interacted with alexa488-labeled PF4 in a concentration-dependent manner, as measured by the quenching of the excitation spectrum. Patients with ant-PF4/heparin antibodies also reacted with LPS/PF4 complex in ELISA. Prior absorption of serum with PF4/heparin complex coated on ELISA plates decreased the reactivity of the serum towards PF4/LPS complex (19–46%) in two out of the five patients tested suggesting some were cross-reaction between PF4/Heparin and PF4/LPS complex. Conclusions PF4 forms a complex with lipopolysaccharide and this complex is immunogenic. Antibodies to PF4/LPS complex can cross-react with PF4/heparin complex raising the possibility that these antibodies may be responsible for the detection of PF4/heparin in individuals never been exposed to heparin previously. These antibodies may also be at least partly responsible for increased thrombosis associated with infection. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet Factor 4 Induces Leukocyte Responses through Integrin Mac-1 (CD11b/CD18)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2529-2529
Author(s):  
Nataly P. Podolnikova ◽  
Valentin P. Yakubenko ◽  
Tatiana P. Ugarova
Keyword(s):  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Structural Similarity ◽  
Peptide Library ◽  
Platelet Factor 4 ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Platelet Factor

Abstract The proteins/peptides secreted from α-granules of activated platelets not only aid in thrombus formation and blood coagulation, but also exert various immune-modulating effects. Among many secreted products, Platelet Factor 4 (PF4), a chemokine that belongs to the CXC family, is one of the most abundant platelet proteins. While PF4 assignment to the chemokine family is based on its structural similarity with other CXC chemokines and chemotactic activity, to date no receptor for PF4 on leukocytes has been identified. Our recent elucidation of the recognition specificity of a major leukocyte integrin αMβ2 (Mac-1) allowed the prediction that PF4 contains several putative Mac-1 recognition motifs and thus could potentially interact with this receptor. Using a peptide library spanning the sequence of PF4, we showed that the αMI-domain of Mac-1 bound several overlapping PF4-derived peptides. The biolayer interferometry analyses demonstrated that PF4 bound recombinant active αMI-domain of Mac-1 in a concentration-dependent manner with a KD of 1.3 ± 0.2x10-6 M. No interaction of PF4 with the inactive αMI-domain (α7 helix extended) or the αLI-domain of a homologous integrin αLβ2 was detected. The full-length recombinant PF4 and the αMI-domain-binding peptide (residues 58-70) identified in the peptide library supported strong adhesion and spreading of Mac-1-expressing cells, including neutrophils, U937 monocytic and Mac-1-transfected HEK293 cells. The cell adhesion to PF4 was partially inhibited by anti-Mac-1 mAbs and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface proteoglycans act cooperatively with integrin Mac-1. PF4 induced a potent migratory response of wild-type, but not Mac-1-deficient, macrophages in a Transwell system. PF4 also enhanced phagocytosis: coating of E. coli bacteria or latex beads with PF4 enhanced ~ 4-fold their phagocytosis by macrophages, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by wild-type, but not Mac-1-deficient, macrophages. These results identify PF4 as a ligand for integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated via interaction with Mac-1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals High-yield production of recombinant platelet factor 4 by harnessing and honing the gram-negative bacterial secretory apparatus

PLoS ONE ◽  
2020 ◽  
Vol 15 (5) ◽  
pp. e0232661
Author(s):  
Saeed Ataei ◽  
Mohammad Naser Taheri ◽  
Gholamhossein Tamaddon ◽  
Abbas Behzad-Behbahani ◽  
Fatemeh Taheri ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
High Yield ◽  
Platelet Factor ◽  
Gram Negative ◽  
Yield Production ◽  
High Yield Production ◽  
Secretory Apparatus

scholarly journals Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment

2016 ◽  
Vol 32 (7) ◽  
pp. 705-717 ◽  
Author(s):  
Zahra F. Parker ◽  
Ann H. Rux ◽  
Amber M. Riblett ◽  
Fang-Hua Lee ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Viral Attachment ◽  
Hiv 1

The Platelet Specific Chemokine Platelet Factor 4 Induces Expression of E-Selectin in an NF-κ B Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3908-3908
Author(s):  
Bruce S. Sachais ◽  
Peihong Ma ◽  
Ann H. Rux ◽  
Guangyao Yu
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Atherosclerotic Lesion ◽  
Surface Expression ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Platelet Factor ◽  
Umbilical Vein Endothelial Cells ◽  
Electrophoretic Mobility Shift Assays

Abstract The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4) is a platelet specific chemokine released upon platelet activation. PF4 has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin mRNA is time and dose dependent, and requires the presence of cell surface glycosaminoglycans. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. Activation of NF-κB is critical for PF4 induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. In summary, our data demonstrate that PF4 can increase expression of E-selectin by endothelial cells by activation of NF-κB. PF4 induction of endothelial E-selectin expression represents another mechanism by which platelets may participate in atherosclerotic lesion progression. These data also suggest that PF4 may participate in the proinflammatory functions of activated platelets.

Disruption of PF4/Hep Multimolecular Complex Assembly Using a Minimally Anticoagulant Heparin (ODSH)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 723-723
Author(s):  
Manali Joglekar ◽  
Pedro Quintana ◽  
Stephen Marcus ◽  
Jian Liu ◽  
Gowthami M. Arepally
Keyword(s):  
Complex Formation ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Antibody Binding ◽  
Fixed Amount ◽  
Platelet Factor ◽  
Molar Ratios

Abstract Abstract 723 Recent studies indicate that multimolecular complexes of platelet factor 4 (PF4) and heparin (H) are central to the pathogenesis of Heparin-Induced Thrombocytopenia (HIT). PF4/H multimolecular complexes are recognized preferentially by HIT antibodies (Rauova, Blood 2005) and are potently immunizing in a murine immunization model (Suvarna, Blood 2005). Because PF4/H multimolecular complexes assemble through non-specific electrostatic interactions, we hypothesized that disruption of PF4/H charge-dependent interactions could reduce immune mediated complications. To test this hypothesis, we employed a minimally anticoagulant compound (2-O, 3-O desulfated heparin, or ODSH, ParinGenix, Inc.) and characterized the charge-dependent interactions of murine PF4 (mPF4), ODSH and unfractionated heparin (UFH). In chromogenic assays of thrombin (IIa) generation, UFH was >80-fold more potent than ODSH in inactivating heparin (IC50 of residual IIa generation for UFH=3.1 nM v. ODSH= 259 nM, (Figure 1A). However, when equimolar amounts of UFH or ODSH (1.7 mM) were tested in a PF4 neutralization assay (Saggin, Thrombosis and Haemostasis 1992), the amount of mPF4 required to neutralize 50% of the anticoagulant activity of ODSH (IC50) was 25μg/mL, as compared to 73μg/mL for UFH (~3-fold difference), indicating that charge-dependent interactions, but not anticoagulant activity, were preserved between PF4 and ODSH (Figure 1B). When ODSH was added at 2.5, 5 or 10 fold molar excess to a fixed amount of UFH (6nM) in the PF4 neutralization assay, a proportionate increase in the amount of PF4 was needed to neutralize UFH, indicating that ODSH promotes the anticoagulant effect of UFH through preferential binding of PF4. To further characterize the biophysical interactions of PF4, ODSH and UFH, we used spectrophotometry and zeta potential to study the multimolecular complex formation (Suvarna, Blood 2007). We noted that mPF4 and ODSH formed multimolecular complexes at molar ratios of 2:1, whereas mPF4 and UFH complexes occurred at molar ratios of 1:1. When increasing concentrations of ODSH were added to pre-formed PF4/H multimolecular complexes, we noted a decrease in absorbance with increasing amounts of ODSH, indicating disruption of PF4/H multimolecular complexes (Figure 1C). However, when increasing amounts of UFH was added to preformed PF4/ODSH multimolecular complexes, a plateau in signal was noted, suggesting a higher affinity of ODSH for PF4. In PF4/H immunoassays, incubation of ODSH (1μg/mL) with HIT antibodies was effective in reducing antibody binding by >50% as compared to wells without ODSH. HIT antibodies did not recognize hPF4 (10mg/mL) in complex with ODSH (0.4-3.2 mg/mL), indicating minimal cross-reactivity of HIT antibodies with PF4/ODSH complexes (Figure 1D). In summary, we show that ODSH, a minimally anticoagulant heparin, can disrupt PF4/H multimolecular complex formation through charge dependent interactions and interfere with HIT antibody binding. These studies suggest that manipulation of PF4:H charge interactions can be a potential therapeutic strategy in the management of HIT. Disclosures: No relevant conflicts of interest to declare.

Histones, Like Platelet Factor 4 (PF4), Affect Generation of Activated Protein C: Implications for the Pathogenesis of Severe Sepsis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 530-530
Author(s):  
M. Anna Kowalska ◽  
Guohua Zhao ◽  
George David ◽  
Mortimer Poncz
Keyword(s):  
Severe Sepsis ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Platelet Factor 4 ◽  
Endothelial Protein C Receptor ◽  
Maximal Increase ◽  
Platelet Factor ◽  
Positively Charged ◽  
Formation Of Complexes

Abstract Abstract 530 Platelet factor 4 (PF4) increases aPC generation by the thrombin (IIa)/thrombomodulin (TM) complex and may impact outcome in sepsis. PF4's effect on aPC generation follows a biphasic curve when tested in solution, on human TM expressing HEK293, and on primary endothelial cells (ECs) with a peak concentration at around 25 μg/ml. Formation of complexes at a specific molar ratio between positively-charged tetramers of PF4 and negatively-charged chondroitin sulfate (CS) on the TM glycosaminoglycan (GAG) is crucial for the increase in aPC generation. Other positively-charged molecules like protamine sulfate (PRT) affect aPC generation in a similar manner, and heparin, which is known to bind PF4 and PRT more avidly than CS, lowers effective PF4 or PRT concentrations. Here we examined whether histones, that are also small positively-charged molecules, affect aPC generation. Histones released from cells in sepsis are cytotoxic toward ECs and lethal when injected into mice, and aPC reverses this lethality. May histones affect aPC generation by the same mechanism as other positively-charged molecules, and how does the presence of PF4 or heparin influence this effect? We have addressed these questions both in solution and with TM-expressing cells, in the absence or presence of endothelial protein C receptor. We found that individual, or mixed histones affect aPC formation following a similar biphasic curve seen with PF4 with a peak effect at around 10 μg/ml but to lesser extent (2-fold maximal increase compared to 6-fold for PF4). Histones and PF4 are additive at low concentrations; however, more importantly, histones only decreased aPC generation when tested in the presence of optimal or higher PF4 concentration (>25 μg/ml). Just as with PF4, added heparin decreased effective histone concentration and shifted the curve for aPC generation to the right, both in the absence or presence of PF4. We hypothesize that normally PF4 released from platelets augments aPC generation and low concentration of histones have similar effect. But when histones are released in sepsis in high concentrations, their interaction with CS on TM blocks formation of complexes between PF4 and TM's CS that are optimal for maximal increase of aPC generation. Further we tested the effect of histones on aPC generation in vivo. Injection of histones in mice increased IIa-induced (2U/kg) aPC generation in plasma. This increase was concentration dependent (at 1 to 20 mg/kg increasing aPC generation up to 10-fold), but injection of higher amount of histones (40 mg/kg) became lethal. Mice that were overexpressing human PF4 had an increased lethality when histones at 40 mg/kg were co-injected with thrombin (2U/kg) over the littermate mice deficient in murine PF4 (60% vs. 0% mortality, respectively, n=5 for each group) suggesting that in vivo histones may also act additively with PF4 on aPC generation. We propose that in severe septic patients, especially those with high levels of released PF4, concurrently available histones suppress aPC generation. By binding to the excess of PF4 and/or histones, heparin may be beneficial in severe sepsis by allowing improved aPC generation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti–platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3791-3796 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Richard J. Cook ◽  
Victor J. Marder ◽  
Jo-Ann I. Sheppard ◽  
Jane C. Moore ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Igg Antibodies ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Antithrombotic Prophylaxis ◽  
High Concentrations ◽  
To Receive

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4) bound to heparin. Immunogenicity of heparins differs in that unfractionated heparin (UFH) induces more anti–PF4/heparin antibodies than low-molecular-weight heparin (LMWH) and UFH also causes more HIT. Fondaparinux, a synthetic anticoagulant modeled after the antithrombin-binding pentasaccharide, is believed to be nonimmunogenic. We tested 2726 patients for anti–PF4/heparin antibodies after they were randomized to receive antithrombotic prophylaxis with fondaparinux or LMWH (enoxaparin) following hip or knee surgery. We also evaluated in vitro cross-reactivity of the IgG antibodies generated against PF4 in the presence of UFH, LMWH, danaparoid, or fondaparinux. We found that anti–PF4/heparin antibodies were generated at similar frequencies in patients treated with fondaparinux or enoxaparin. Although antibodies reacted equally well in vitro against PF4/UFH and PF4/LMWH, and sometimes weakly against PF4/danaparoid, none reacted against PF4/fondaparinux, including even those sera obtained from patients who formed antibodies during fondaparinux treatment. At high concentrations, however, fondaparinux inhibited binding of HIT antibodies to PF4/polysaccharide, indicating that PF4/fondaparinux interactions occur. No patient developed HIT. We conclude that despite similar immunogenicity of fondaparinux and LMWH, PF4/fondaparinux, but not PF4/LMWH, is recognized poorly by the antibodies generated, suggesting that the risk of HIT with fondaparinux likely is very low.

scholarly journals Endothelial expression of E-selectin is induced by the platelet-specific chemokine platelet factor 4 through LRP in an NF-κB–dependent manner

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3545-3551 ◽  
Author(s):  
Guangyao Yu ◽  
Ann H. Rux ◽  
Peihong Ma ◽  
Khalil Bdeir ◽  
Bruce S. Sachais
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Nuclear Factor Κb ◽  
Platelet Factor 4 ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Platelet Factor

AbstractThe involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4), a platelet-specific chemokine released on platelet activation, has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin RNA is time and dose dependent. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. PF4 induces E-selectin expression by activation of transcriptional activity. Activation of nuclear factor-κB is critical for PF4-induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. Further, we have identified the low-density lipoprotein receptor-related protein as the cell surface receptor mediating this effect. These results demonstrate that PF4 is able to increase expression of E-selectin by endothelial cells and represents another potential mechanism by which platelets may participate in atherosclerotic lesion progression.

scholarly journals Studies of thromboxane B2, platelet factor 4, and fibrinopeptide A in bleeding-time blood of patients deficient in von Willebrand factor, platelet glycoproteins Ib and IIb-IIIa, and storage granules

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 481-490 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Bleeding Time ◽  
Platelet Factor 4 ◽  
Thromboxane B2 ◽  
Initial Slope ◽  
Dependent Manner ◽  
High Shear ◽  
Fibrinopeptide A ◽  
Wound Surface ◽  
Platelet Factor ◽  
Fibrin Formation

The blood volumes and concentrations of thromboxane B2 (TxB2), platelet factor 4 (PF4), and fibrinopeptide A (FPA) were measured every 30 seconds in bleeding-time blood in normal subjects and in patients with idiopathic thrombocytopenic purpura (ITP), delta and alpha delta storage pool deficiency (SPD), Bernard-Soulier Syndrome (BSS), thrombasthenia (TSA), and von Willebrand's disease (vWD). Data were fitted to second-order (TxB2, PF4, and FPA) or third-order (volumes) polynomials. Average values for various parameters over fixed-time intervals were determined by numerical methods. The bleeding time was greater than 15 minutes in all patient groups and the initial bleeding, as reflected by the initial slope of the fitted blood volume curves, was increased in ITP, BSS, and SPD (delta-SPD in particular), but not in vWD and TSA. The increased values for both the initial slope and the volume of blood collected after 2 minutes in SPD suggest that vascular tone may be modulated, in part, by dense granule substances such as adenosine triphosphate (ATP) or serotonin. In TSA, uniquely, both platelet (TxB2 and PF4) and coagulation (FPA) values were increased in early bleeding samples (initial slope). In vitro studies of TxB2 production, together with previous flow studies of fibrin formation, also suggest enhanced activation and coagulant properties of thrombasthenic platelets. In other patients, reduced values of all substances at later times may reflect impaired platelet-fibrin plug formation in the high-shear regions at the ends of transected blood vessels. However, the initial slopes of the fitted curves for both TxB2 and PF4 were normal in vWD, suggesting that the early appearance of these substances may typically be from platelets that are adherent to collagen within the lower shear environment of the wound surface. The finding that FPA values were not decreased initially in any patient group, including ITP, but were decreased at later times (except for TSA), suggests that early fibrin formation occurs independently of platelets in the low-shear environment of the wound surface, whereas at later times fibrin is formed in a platelet-dependent manner in the high- shear regions at the ends of transected vessels.

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