Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

High prevalence of antibodies to platelet factor 4 heparin in patients with antiphospholipid antibodies in absence of heparin-induced thrombocytopenia

Seventy-two patients with antiphospholipid antibodies (aPL), with or without antiphospholipid syndrome (APS), were studied for detection of heparin-PF4-induced antibodies (HPIA) using a commercial kit (Asserachrom HPIA) PF4-dependant enzyme-linked immunoassay (ELISA) test. None of the patients had a medical history of heparin induced thrombocytopenia (HIT). Eleven percent of patients were positive for HPIA. Plasma from 40 of the 72 patients (seven positive and 33 negative), was also tested with the other available HPIA ELISA (GTI) kit. Five patients were positive with both ELISA kits, two were highly positive only with Asserachrom HPIA and four only with GTI. None of the positive patients had severe thrombocytopenia. Two patients have never received heparin treatment. No relationship was found between HPIA presence and patients' age, sex, aPL levels or presence of lupus anticoagulant. No significant difference in HPIA presence was observed in patients with primary APS, secondary APS or aPL without APS. We found a poor correlation between the two commercial ELISA showing that, on the same blood sample, a patient could be highly positive with one technique and negative with the other. The PF4-dependant enzyme-linked immunoassay, which is often the first test used for the diagnosis of HIT, should be interpreted cautiously in patients with aPL since there is a danger of overdiagnosis and overtreatment.     

Induction of monocyte tissue factor expression by antibodies to heparin-platelet factor 4 complexes developed in heparin-induced thrombocytopenia

The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xa-based chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients.     

The complicated relationships of heparin-induced thrombocytopenia and platelet factor 4 antibodies with COVID-19

COVID-19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID-19-infected patients. This has thus been identified in some publications as representing a high incidence of heparin-induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin-induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin-dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID-19-infected patients, and they may occur at higher incidence than in historical non-COVID-19-infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin-dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT-like” syndrome has reported in patients who have been vaccinated against COVID-19. Accordingly, much more is yet to be learnt about the insidious disease that COVID-19 represents, including autoimmune outcomes in affected patients.     

The homozygous FcgammaRIIIa-158V genotype is a risk factor for heparin-induced thrombocytopenia in patients with antibodies to heparin-platelet factor 4 complexes

We hypothesized that Fcgamma receptor IIIa (FcgammaRIIIa), a polymorphic receptor for the Fc portion of immunoglobulin G (IgG) other than FcgammaRIIa, was involved in heparin-induced thrombocytopenia (HIT). FcgammaRIIa-131 and FcgammaRIIIa-158 genotypes were determined in 102 patients with definite HIT and in 2 control groups of patients treated by heparin (86 subjects without detectable antibodies [Abs] to heparin-platelet factor 4 [H/PF4], Ab(-) group; 84 patients with Abs to H/PF4 without HIT, Ab(+) group). There were no significant differences in genotype distribution or allele frequencies between the 3 groups for FcgammaRIIa-131H/R polymorphism. In contrast, FcgammaRIIIa-158V homozygotes were more frequent in the HIT group than in the Ab(+) group (P = .02), a difference that was more pronounced in patients with high levels of anti-H/PF4 Abs (P = .01). Since anti-H/PF4 Abs are mainly IgG1 and IgG3, clearance of sensitized platelets may be increased in patients homozygous for the FcgammaRIIIa-158V allotype, thus contributing to the development of thrombocytopenia.     

Incidence of antiheparin-platelet factor 4 antibodies and heparin-induced thrombocytopenia in Turkish patients undergoing cardiac surgery.

The frequency of antiheparin-platelet factor 4 antibodies by means of antigenic and functional assays ((14) C-serotonin release assay and citrated plasma platelet aggregation) was determined in 115 Turkish patients undergoing cardiac surgery. Blood samples were taken immediately before surgery and on days 5 and 10 +/- 2. Platelet counts were recorded and thrombotic events were determined by clinical methods. Antibody generation measured by enzyme-linked immunosorbent assay before surgery (n = 44) and on days 5 (n = 44) and 10 (n = 115) was 15.9%, 34.1%, and 65.2%, respectively. Positive samples from functional assays were 4.4% on day 0 and 7.0% on day 10. All positive samples had been negative on day 0. A high frequency of antiheparin-platelet factor 4 antibody generation and a low frequency of clinical heparin-induced thrombocytopenia were determined in these patients. These results obtained for Turkish patients are similar to those of other studies of heparin-induced thrombocytopenia.     

The role of platelet factor 4 in platelet aggregation induced by the antibodies implicated in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment. Recent studies using immunological methods demonstrated that antibodies contained in plasma, or in purified total immunoglobulin (Ig)G from patients suffering HIT, recognize as target antigen the complex heparin/platelet factor (PF4). In the present study, the role of PF4 in in-vitro platelet aggregation induced by purified total IgG or platelet-poor plasma from patients suffering HIT was investigated. In order to demonstrate the functional role of PF4, an anti-PF4 antibody that specifically blocked PF4 was used. In an experimental system composed of washed platelet suspension, incubation of F(ab')2 fragments (0.125 microg/ml) of the polyclonal anti-PF4 antibody resulted in complete inhibition of platelet aggregation triggered by purified total IgG from patients suffering HIT and heparin. In platelet-rich plasma, a significantly higher concentration (4.25 microg/ml) of the anti-PF4 F(ab')2 was required to inhibit platelet aggregation induced by HIT-PPP and heparin. Intermediate concentrations of the anti-PF4 antibody partially inhibited platelet aggregation. In plasma milieu, the concentration of PF4 was about five-fold higher in comparison with that measured in the purified system. The intensity of platelet aggregation depended on the concentration of HIT-IgG. Platelet aggregation was abolished in the presence of high concentrations of heparin (superior or equal to 10 IU/ml). The present study shows that PF4 is essential for platelet aggregation triggered by the antibodies related to HIT in the presence of heparin. The concentration of PF4 that is available to bind with heparin or with the HIT-related antibodies is critical for platelet aggregation induced by HIT antibodies.     

Platelet response to direct thrombin inhibitor or fondaparinux treatment in patients with suspected heparin-induced thrombocytopenia

Making a definitive diagnosis of heparin-induced thrombocytopenia (HIT) can be problematic. A prompt platelet rise following treatment has been proposed as a “post-test” criterion for diagnosis. However, the platelet response following discontinuation of heparin and initiation of a recommended alternative anticoagulant remains largely undefined and unstudied. This study aimed to characterize platelet response to initial treatment in patients with a low, intermediate, or high likelihood of having HIT. This was a multicenter retrospective cohort study. Patients were over 18 years in age, underwent serologic testing for HIT, and received alternative anticoagulation treatment for HIT. Classification of each patient’s likelihood of having HIT was based on an empiric, pre-hoc combination of the 4T score and serology results. The primary outcome for this study was a platelet count response after initiation of direct thrombin inhibitor (DTI) or fondaparinux therapy within 48 h. 124 patients were analyzed. The sensitivity and specificity of having an immediate platelet rise of at least 10,000/µL by day 2 after starting treatment among high-likelihood for HIT patients were 0.71 (95% CI 0.55–0.84) and 0.64 (95% CI 0.5–0.76), respectively. The negative predictive value of no platelet rise was 75.5% (95% CI 0.61–0.86). A prompt platelet count rise may be appropriate to consider along with other known criteria for the clinical diagnosis of HIT. The rise should be immediate following discontinuation of heparin and initiation of recommended treatment, with an upward rise within 48 h.     

Functional characterization of antibodies against heparin-platelet factor 4 complex in heparin-induced thrombocytopenia patients in Asian-Indians: relevance to inflammatory markers.

Occurrence of heparin-induced thrombocytopenia (HIT) was investigated for 33 Indian patients undergoing cardiovascular surgery who received unfractionated heparin (UFH). Platelet counts were performed prior to the initiation of UFH therapy and 5-16 days post therapy. Heparin-induced platelet aggregation, C-serotonin release assay, and enzyme-linked immmunosorbent assay (ELISA) tests were performed in all the patients to detect the antibodies formed against the complex of heparin and platelet factor 4 (HPF4). Levels of inflammatory markers/mediators such as CD40 ligand (CD40L) and C-reactive protein (CRP) were also measured in the patient plasmas utilizing ELISA tests. Based on clinical observations and laboratory diagnoses, five patients (15%) were considered to have confirmed HIT. Despite wide variations in the titers of inflammatory markers, patients who tested ELISA-positive for HPF4 antibodies showed markedly elevated levels of both soluble CD40L and C-reactive protein. Most strikingly, the C-serotonin release assay-positive patients showed up to a 10-fold increase in the level of CD40L. It is concluded that approximately 15% Asian-Indian patients receiving UFH during cardiovascular surgery develop functional HPF4 antibodies, which are associated with the increased levels of inflammatory markers/mediators in this catastrophic HIT syndrome.     

Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified from the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was obtained using 0.1 M glycine/0. 5 M NaCl at pH 2.5 (peak 2). In an enzyme-linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H-PF4 complexes. Finally, IgG in patient 3 only bound to H-PF4 and was unreactive with PF4 alone. Using [14C]-serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.     

Investigation of a platelet factor 4 polymorphism on the immune response in patients with heparin-induced thrombocytopenia

A small fraction of patients who receive heparin develop heparin-induced thrombocytopenia (HIT) and, of these patients, a still smaller proportion develop associated thrombotic complications. Heparin-induced thrombocytopenia is caused by the formation of antibodies that bind to specific complexes of platelet factor 4 (PF4) and heparin. However, it remains uncertain why certain patients form these antibodies and develop HIT or why certain patients have thrombotic events. In this report we describe studies on individuals with and without HIT to determined if a potential PF4 polymorphism could explain differences in susceptibility to HIT. In the 10 control individuals and the 10 patients we studied, we did not find a difference in the PF4 sequences. Genetic difference in the PF4 antigenic target does not explain the occurrence of HIT in susceptible patients.     

Investigation of a platelet factor 4 polymorphism on the immune response in patients with heparin-induced thrombocytopenia

A small fraction of patients who receive heparin develop heparin-induced thrombocytopenia (HIT) and, of these patients, a still smaller proportion develop associated thrombotic complications. Heparin-induced thrombocytopenia is caused by the formation of antibodies that bind to specific complexes of platelet factor 4 (PF4) and heparin. However, it remains uncertain why certain patients form these antibodies and develop HIT or why certain patients have thrombotic events. In this report we describe studies on individuals with and without HIT to determined if a potential PF4 polymorphism could explain differences in susceptibility to HIT. In the 10 control individuals and the 10 patients we studied, we did not find a difference in the PF4 sequences. Genetic difference in the PF4 antigenic target does not explain the occurrence of HIT in susceptible patients.     

Pathogenicity of IgA and/or IgM antibodies to heparin–PF4 complexes in patients with heparin-induced thrombocytopenia

Antibodies to heparin–PF4 (H-PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin-induced thrombocytopenia (type II HIT). All the patients had a platelet count < 120 × 10⁹/l or a reduction of >30% of the initial value, occurring at least 5 d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases ( n  = 26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and/or IgM anti-H-PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcγRII) are not necessarily involved in the pathogenicity of heparin-dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H-PF4 on the platelet surface, may directly trigger platelet activation, aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcαR) or IgM (FcμR). IgM–platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans–PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis.     

Pathogenicity of IgA and/or IgM antibodies to heparin–PF4 complexes in patients with heparin-induced thrombocytopenia

Antibodies to heparin–PF4 (H-PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin-induced thrombocytopenia (type II HIT). All the patients had a platelet count < 120 × 10⁹/l or a reduction of >30% of the initial value, occurring at least 5 d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases (n = 26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and/or IgM anti-H-PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcγRII) are not necessarily involved in the pathogenicity of heparin-dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H-PF4 on the platelet surface, may directly trigger platelet activation, aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcαR) or IgM (FcμR). IgM–platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans–PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis.     

Platelet aggregation in response to four low molecular weight heparins and the heparinoid ORG 10172 in patients with heparin-induced thrombocytopenia

Summary A simple rapid platelet aggregation test was used to evaluate cross-reactivity of four low molecular weight heparins and the heparinoid ORG 10172 in three patients with heparin-induced thrombocytopenia. The low molecular weight heparins cross-reacted in 11 out of 12 tests. The heparinoid ORG 10172 did not cross-react in any of the patients. One patient was treated with ORG 10172 and thrombocytopenia resolved.     

Rapid determination of anti-heparin/platelet factor 4 antibody titers in the diagnosis of heparin-induced thrombocytopenia

PURPOSE: Heparin-induced thrombocytopenia is mediated by antibodies directed against the heparin-platelet factor 4 (heparin/PF4) complex. Our aim was to investigate whether rapid measurement of anti-heparin/PF4 antibodies could improve the diagnostic workup of patients with suspected heparin-induced thrombocytopenia. METHODS: We examined 148 consecutive patients in our laboratory between January 1995 and June 2001 for suspected heparin-induced thrombocytopenia. Clinical data allowed retrospective assessment of the likelihood of heparin-induced thrombocytopenia. Antibodies against the heparin/PF4 complex were detected by a rapid particle gel immunoassay. RESULTS: Anti-heparin/PF4 antibodies were detected in 69 (47%) of the 148 patients, at dilution titers from 1 to 256. Clinically "likely" or "very likely" heparin-induced thrombocytopenia was significantly more common in patients with titers >or=4 (95% [39/41]) than in those with undetectable antibodies (13% [9/70]; P <0.0001), a titer of 1 (18% [4/22]; P <0.0001), or a titer of 2 (33% [2/6]; P = 0.001). All 19 samples with a positive platelet aggregation test had anti-heparin/PF4 antibody titers of at least 4, including 15 samples with titers >or=32. Thromboembolic complications in heparin-treated patients were significantly more prevalent in patients with titers >or=4 (63% [26/41]) than in those with undetectable antibodies (8% [6/79]; P <0.0001) or a titer of 1 (9% [2/22]; P <0.0001). Of the 11 patients with a titer of 1 who were maintained on heparin, none developed worse thrombocytopenia or thromboembolic complications. CONCLUSION: Anti-heparin/PF4 antibody titers, which can be measured rapidly and reproducibly using a particle gel immunoassay, can be used as a confirmatory test to complement a clinical likelihood score among patients with suspected heparin-induced thrombocytopenia.     

SET DOMAIN GROUP2 is the major histone H3 lysine [corrected] 4 trimethyltransferase in Arabidopsis

Posttranslational modifications of histones play important roles in modulating chromatin structure and regulating gene expression. We have previously shown that more than two thirds of Arabidopsis genes contain histone H3 methylation at lysine 4 (H3K4me) and that trimethylation of H3K4 (H3K4me3) is preferentially located at actively transcribed genes. In addition, several Arabidopsis mutants with locus-specific loss of H3K4me have been found to display various developmental abnormalities. These findings suggest that H3K4me3 may play important roles in maintaining the normal expression of a large number of genes. However, the major enzyme(s) responsible for H3K4me3 has yet to be identified in plants, making it difficult to address questions regarding the mechanisms and functions of H3K4me3. Here we described the characterization of SET DOMAIN GROUP 2 (SDG2), a large Arabidopsis protein containing a histone lysine methyltransferase domain. We found that SDG2 homologs are highly conserved in plants and that the Arabidopsis SDG2 gene is broadly expressed during development. In addition, the loss of SDG2 leads to severe and pleiotropic phenotypes, as well as the misregulation of a large number of genes. Consistent with our finding that SDG2 is a robust and specific H3K4 methyltransferase in vitro, the loss of SDG2 leads to a drastic decrease in H3K4me3 in vivo. Taken together, these results suggest that SDG2 is the major enzyme responsible for H3K4me3 in Arabidopsis and that SDG2-dependent H3K4m3 is critical for regulating gene expression and plant development.     

Antihirudin antibodies in patients with heparin-induced thrombocytopenia treated with lepirudin: incidence, effects on aPTT, and clinical relevance

Hirudin, a potent and specific thrombin inhibitor, is a protein of nonhuman origin and therefore potentially immunogenic. The primary objectives of this investigation were to determine the incidence of antihirudin antibodies (ahir-ab) in patients with heparin-induced thrombocytopenia (HIT) who received lepirudin as parenteral anticoagulation and to determine the incidence of death, limb amputation, new thromboembolic complications (TECs), and major hemorrhage in patients who had ahir-ab, compared with patients who were ahir-ab negative. The investigation used data from 2 prospective multicenter studies with the same study protocol, in which HIT patients received 1 of 4 intravenous lepirudin dosage regimens. The treatment duration was 2 to 10 days. Ahir-ab were determined by a newly developed enzyme-linked immunosorbent assay (ELISA). Eighty-seven of 196 evaluable patients (44.4%) had ahir-ab of the IgG class. Development of ahir-ab was dependent on the duration of treatment (ahir-ab-positive patients 18.6 days vs ahir-ab-negative patients 11.8 days; P =.0001). Fewer ahir-ab-positive than ahir-ab-negative patients died (P =.001). Ahir-ab did not cause an increase in limb amputation (P =.765), new TECs (P >.99), or major bleedings (P =.549). In 23 of 51 (45.1%) evaluable patients in whom ahir-ab developed during treatment with lepirudin ( = 12% of all lepirudin treated patients), the ahir-ab enhanced the anticoagulatory effect of lepirudin. Ahir-ab are frequent in patients treated with lepirudin for more than 5 days. Ahir-ab are the first example for a drug-induced immune response causing enhanced activity of a drug. Therefore, during prolonged treatment with lepirudin, anticoagulatory activity should be monitored daily to avoid bleeding complications.     

Interplay of platelet polymorphisms, risk factors, and von [corrected] Willebrand factor [corrected] in determining collagen-adenosine diphosphate PFA-100 results in patients with coronary artery disease.

Platelets play a pivotal role in thrombus formation in patients with coronary artery disease (CAD), since the high shear generated in the presence of severe coronary stenoses can increase platelet reactivity (PR) and trigger thrombogenesis. Several reports have suggested a functional effect of human platelet antigen (HPA)-1 and HPA-2 gene polymorphisms on PR. However, the true determinants of high-shear PR in CAD patients taking their usual medications are still incompletely understood. In 104 patients with stable CAD we analyzed the possible clinical, biochemical and genetic factors affecting high-shear PR, measured by the ex vivo platelet function analyzer (PFA-100) collagen-adenosine diphosphate method. In univariate analysis, a lower PR was associated with decreased plasma von Willebrand factor-ristocetin cofactor activity, increased blood levels of triglycerides, female sex, use of thienopyridines, lower platelet count, and HPA-1b carriership. All variables, except HPA-1b, remained associated with lower PR in multivariate analysis. However, the introduction in the model of the HPA-1 and HPA-2 genotypes as interaction terms led to a significant improvement in the prediction of PR, although the quantitative effect was small (about 3% improvement, P=0.046).Thus, in CAD patients, there seems to be only a mild effect of the platelet glycoprotein HPA-1 and HPA-2 polymorphisms on collagen-adenosine diphosphate-stimulated PR after the effect of well-established clinical and biochemical determinants are considered.