Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 2.

Platelet signal transduction is the focus of this review. While 'classic' platelet signaling through G protein-coupled receptors in response to fluid-phase agonists has been extensively studied, signaling mechanisms linking platelet adhesion receptors such as GPIb-IX-V, GPVI and α2ß1 to the activation of αIIbß3 are less well established. Moreover, 'non-haemostatic' pathways can also activate platelets in various settings, including platelet-immune or platelet-tumour cell interactions, platelet responses to neutrophil extracellular traps, or stimulation by microbial pathogens. Genetically determined integrin variants can modulate platelet function and increase thrombogenicity. A typical example is the Pro33 (HPA-1b) variant of αIIbß3. Recent advances in the genotype-phenotype relation of this prothrombotic variant and its impact on outside-in signaling will be reviewed.

Do we need antiplatelet therapy in thrombocytosis? Contra. Proposal for an individualized risk-adapted treatment.

Thrombocytosis is a frequent laboratory finding but not a diagnosis. Therefore, elevated platelet counts (>450 x 109/l) require careful diagnostic work-up to differentiate between reactive thrombocytosis (RT), caused by various conditions, and essential thrombocythemia (ET), a myeloproliferative neoplasm (MPN). In either setting, aspirin is widely used in clinical practice. However, RT (even at platelet counts >1000 x 109/l) has never been shown to cause thrombosis or bleeding due to acquired von Willebrand factor defects in association with high platelet counts. Identification of reactive conditions and appropriate therapy of the underlying disorder are most relevant. By contrast to RT, ET and related MPN can be associated with thrombosis and/or hemorrhage. Current recommendations suggest the use of low-dose aspirin in all patients with ET unless contraindicated. However, the strength of this recommendation is weak, i. e. evidence level IIb grade B. A potential benefit of aspirin used for primary thromboprophylaxis in ET is mostly derived from the ECLAP study in polycythemia vera (PV). However, translating study results from PV to ET appears to be highly questionable and may be biased. In the absence of robust data regarding the benefit-risk balance of aspirin in ET, it appears reasonable (1) to stratify patients according to their individual thrombotic and bleeding risk, (2) to restrict the use of aspirin to high-risk categories and patients with microcirculatory disturbances, (3) to test for pharmacological efficacy (COX-1 inhibition; measurement of TXB2), and (4) to modify the aspirin dosing regimen (twice instead of once daily) if required.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

Laboratory diagnosis of acquired hemophilia A: limitations, consequences, and challenges.

Acquired hemophilia A (AHA) should be suspected in patients with a new onset of bleeding and an isolated prolongation of activated partial thromboplastin time. About 10% of patients do not bleed at the time of diagnosis, but are at risk of future bleeding, particularly during interventions or surgery. Diagnosis of AHA is confirmed by demonstrating markedly reduced factor VIII activity (FVIII:C) and neutralizing anti-FVIII antibodies, so-called inhibitors. Several limitations and pitfalls exist with the assays used to diagnose AHA. Interference can result from anticoagulants or lupus anticoagulant. The Bethesda assay used to measure inhibitor potency assumes a log-linear relationship between inhibitor concentration and effect on residual FVIII:C activity to allow exact quantification. However, this relationship is not present for the type 2 inhibitors typically seen in AHA. Therefore, this assay only provides a rough estimate of inhibitor potency. These limitations can explain, in part, why laboratory data, such as inhibitor potency, failed to predict bleeding or response to treatment in AHA. This article reviews the diagnostic approach to AHA, discusses assay-specific limitations and addresses some of the challenges for future research.

Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

Drugs that affect platelet function.

Drugs represent the most common cause of platelet dysfunction in our overmedicated society. While acetylsalicylic acid (aspirin), adenosine diphosphate receptor antagonists (clopidogrel and prasugrel), and integrin αIIbß3 (GPIIb-IIIa) receptor blockers (abciximab, eptifibatide, and tirofiban) are well-known prototypes of antiplatelet drugs, other widely used agents such as nonsteroidal anti-inflammatory drugs, antibiotics, cardiovascular and lipid-lowering drugs, selective serotonin reuptake inhibitors, and volume expanders can also impair platelet function and thus cause or aggravate hemorrhages in certain clinical settings. Therefore, induction of a bleeding diathesis remains a significant concern. This is especially relevant in patients with preexisting hemostatic defects of any kind, which may remain compensated as long as platelet function (and/or coagulation) is not inhibited pharmacologically. Identification of individual patients with preexisting hemostatic defects remains crucial (1) to prevent otherwise unexpected bleeding complications, (2) to manage hemorrhagic symptoms adequately, (3) to minimize the risk from invasive procedures, and (4) to avoid unnecessary patient exposure to blood products. This article provides a review of the large variety of agents that have not been designed for antiplatelet therapy but nevertheless interfere with platelet reactivity or induce platelet inhibition. In particular, drug interactions and mechanisms by which these agents can trigger or cause platelet dysfunction are detailed.

Fatal bleeding due to a heparin-like anticoagulant in a 37-year-old woman suffering from systemic mastocytosis.

A 37-year-old female patient with systemic mastocytosis who was admitted with severe unexplained bleeding symptoms is studied. Laboratory procedures established the diagnosis of a patient-derived-heparin-like anticoagulant as a very rare hemostatic abnormality predisposing to bleeding. The patient died from refractory disease despite therapy with protamine, initiation of chemotherapy, and supportive measures. The case illustrates the clinical presentation and diagnosis of heparin-like anticoagulants. Etiology, pathophysiology, and therapeutic options are discussed.

Inherited thrombophilia and gestational venous thromboembolism.

Thromboembolic disease remains a leading cause of maternal mortality during pregnancy and the puerperium. Rational and risk-adapted administration of heparin prophylaxis depends on the identification of those women who have an increased risk of thrombosis and the accurate quantification of this risk. In women without prior thrombosis, the presence of a heterozygous factor V Leiden or heterozygous G20210A mutation in the prothrombin gene is associated with a pregnancy-associated thrombotic risk of approximately 1 in 400. Thus, in pregnant carriers of either one of these mutations, the risk of venous thromboembolism is low. Therefore, no heparin prophylaxis is recommended. A combination of the two genetic risk factors can increase the risk to a modest level of 1 in 25. In all women with prior thrombosis, the authors recommend heparin prophylaxis throughout pregnancy and postpartum for 6 weeks (inconsistent data). However, according to the American College of Chest Physicians recommendations, in the subgroup of women with an episode of prior thrombosis associated with a transient risk factor, such as surgery or trauma, and no additional genetic risk factor, clinical surveillance throughout pregnancy and heparin prophylaxis postpartum is possible. Despite the remarkable progress in risk stratification, the absolute magnitude of risk and the optimum management is, in many cases, an issue of ongoing debate.

The screening power of methylenetetrahydrofolate reductase C677T polymorphism versus plasma homocysteine concentration in patients with stenosis of the internal carotid artery.

BACKGROUND: Hyperhomocysteinemia is an important and independent risk factor for vascular disease. About 35% of patients with stroke and 47% of patients with peripheral arterial disease have elevated plasma homocysteine (HCY) concentrations. The relationship between plasma HCY and the methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is still unclear, especially in regard to screening/diagnostic power. METHODS: This case-control study was performed on 96 patients, who underwent surgery due to asymptomatic or symptomatic high grade stenosis of the internal carotid artery (ICA), and 96 healthy age and sex-matched, controls. Plasma HCY concentration was determined using a commercial kit for fully automated analysis (AxSYM, Abbott). The C677T polymorphism of the MTHFR-gene was assessed by PCR. RESULTS: The mean plasma HCY concentration was significantly higher in the group with stenosis of ICA compared to the controls, 12.43 +/- 6.96 microM and 10.16 +/- 3.16 microM, respectively, (p < 0.05). An HCY plasma concentration of 1.5 SD above the mean value of the control group, was defined as cut-off for a pathological versus physiological plasma concentration. The sensitivity and specificity of HCY was 0.27 and 0.94, respectively. The positive predictive value was 0.82. There was no significant difference in the frequency of the MTHFR 677 CT and TT genotype between patients and controls (47% vs. 47% and 8.3% vs. 11.4%, respectively). Carriers of the T-allele (CT and TT genotypes) have significantly higher plasma HCY concentrations than CC patients, 14.1 +/- 7.6 microM and 10.29 +/- 5.2 microM, respectively, p < 0.05. Sensitivity and specificity of the MTHFR C677T polymorphism (T-allele) were 0.56 and 0.40, respectively. The positive predictive value was 0.48. There was no significant difference in plasma HCY or genotype frequency of the MTHFR C677T polymorphism between asymptomatic and symptomatic patients. CONCLUSION: Our study shows that in a population with a given pretest disease probability of 50%, the determination of plasma HCY concentration, with a positive predictive value of 0.82, is more suitable for screening of patients at risk than analysis of the MTHFR C677T polymorphism.

Maternal IVS1-401 T allele of the estrogen receptor alpha is an independent predictor of late fetal loss.

OBJECTIVE: To investigate whether sequence variants in the gene encoding for estrogen receptor alpha (ER-alpha) are risk determinants for fetal loss. DESIGN: Case-control study. SETTING: University medical center. PATIENT(S): One hundred four women with a history of fetal loss and 277 healthy women with at least one previous pregnancy and no previous fetal loss. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The IVS1-401C/T polymorphism of the human ER-alpha, the G1691A mutation of the factor V gene (factor V Leiden), the G20210A mutation of the prothrombin gene, and the C677T polymorphism of the methylenetetrahydrofolate-reductase (MTHFR) gene were determined by polymerase chain reaction. RESULT(S): In the subgroup analysis of women with at least one late miscarriage (n = 70), the prevalences of the ER-alpha IVS1-401 T allele (T/T vs. C/C, odds ratio [OR]: 2.85, P=.018; T/T + C/T vs. C/C, OR: 2.28, P=.043) and of heterozygous factor V Leiden (OR, 3.2; P=.002) were significantly higher among women with late fetal loss than among healthy women. Carriers of both risk determinants have an at-least additive increase in risk for late abortions (OR, 7.0; P=.0004). The population of all late abortions that would be attributable to the genetic variants (population attributable risk) was 13.9% for factor V Leiden and 49.2% for the ER-alpha IVS1-401 T allele. CONCLUSION(S): Women with the IVS1-401 T allele of the ER-alpha and/or factor V Leiden are at increased risk for late fetal loss.

Inherited thrombophilia and gestational venous thromboembolism.

Thromboembolic disease is a leading cause of maternal morbidity and mortality during pregnancy and the puerperium. To reduce the incidence of venous thromboembolism in pregnancy and improve outcomes, an individual risk stratification based on probability of thrombosis as a rationale for an individual risk-adapted prophylaxis is required. Within the past 10 years, a significant improvement in risk estimation has been achieved due to the identification of new genetic risk factors of thrombosis. In women without prior thrombosis, the presence of a heterozygous factor V Leiden or heterozygous G20210A mutation in the prothrombin gene is associated with a pregnancy-associated thrombotic risk of approximately 1 in 400. Thus, in pregnant carriers of either one of these mutations the risk of venous thromboembolism is low--indicating that pregnancy-associated thrombosis is multicausal, resulting from the interaction of combined defects. A combination of the two genetic risk factors can increase the risk to a modest level (risk 1 in 25). In women with a single episode of prior thrombosis associated with a transient risk factor, for example, surgery or trauma, and no additional genetic risk factor, the probability of a pregnancy-associated thrombosis appears also to be low. In contrast, in women with a prior idiopathic venous thrombosis who carry an additional hereditary risk factor or who have a positive family history of thrombosis, a high risk (>10%) can be expected, supporting the indication for active antepartum and postpartum heparin prophylaxis. Despite the remarkable progress in risk stratification, the absolute magnitude of risk in many cases is unknown and current recommendations remain empirical.

Prediction, prevention, and treatment of venous thromboembolic disease in pregnancy.

Pregnancy is recognized as an independent risk factor for venous thromboembolism leading to thromboembolic events, particularly in women with prior venous thrombosis, family history of thrombosis, or additional thrombophilic risk factors. To reduce the incidence of venous thromboembolism in pregnancy and improve outcomes, an individual risk stratification on the basis of probability of thrombosis as a rationale for an individual risk-adapted prophylaxis is required. In women without prior thrombosis, the presence of a heterozygous factor V Leiden or heterozygous G20210A mutation in the prothrombin gene is associated with a pregnancy-associated thrombotic risk of approximately 1 in 400. Thus, in pregnant carriers of either one of these mutations the risk of venous thromboembolism is low. For this reason, routine thrombophilia screening of all pregnant women is not recommended. However, a combination of the two genetic risk factors can increase the risk to a modest level (risk 1 in 25). In women with a single episode of prior thrombosis associated with a transient risk factor (such as surgery or trauma) and no additional genetic risk factor, the probability of a pregnancy-associated thrombosis also appears to be low. In contrast, in women with a prior idiopathic venous thrombosis who carry an additional hereditary risk factor or who have a positive family history of thrombosis, a high risk (> 10%) can be expected, supporting the indication for active antepartum and postpartum heparin prophylaxis. In many cases, the absolute magnitude of risk is unknown or estimated, and recommendations are often empiric.

Deep venous thrombosis during pregnancy and after delivery: indications for and results of thrombectomy.

PURPOSE: Pregnancy and the puerperium are time periods of an increased risk for venous thromboembolism. An ideal treatment should lead to complete restoration of the venous lumen, elimination of the embolic source, and prevention of severe postphlebitic syndrome. Anticoagulation therapy with heparin or thrombectomy are treatment options. In the current literature, these options are discussed controversially. METHODS: From January 1982 to December 2001, 97 women underwent (93% transfemoral) thrombectomy and construction of an arteriovenous fistula (AVF) for deep venous thrombosis related to pregnancy. The clinical and coagulation parameters were evaluated. The AVF was ligated 3 to 6 months later. Follow-up with duplex ultrasound scan, photoplethysmography, and strain-gauge plethysmography was completed in 87 women. RESULTS: Surgery was performed without any maternal death or pulmonary embolization. A cesarean section was carried out during the same anesthesia in 11 cases. Thrombectomy was completed with construction of a temporary AVF in 90 patients (92.8%). One fetal death occurred in the recovery room for unknown reasons. In the early postoperative course, 16 patients (16.5%) underwent redo surgery for rethrombosis with or without the occlusion of the fistula. In 14 of these patients, the venous system remained patent thereafter. Fetal or neonatal death occurred in five cases 2 to 10 weeks after surgery, mainly because of abruption of the placenta probably from anticoagulation. Among 247 preoperatively occluded anatomic regions, 221 could be restored, and the secondary patency rate amounted to 89.5%. After a mean follow-up period of 6 years, 49 patients (56.3%) were seen without a postphlebitic syndrome, and only three patients (3.5%) had had a leg ulcer develop. CONCLUSION: In experienced hands, venous thrombectomy is a safe method to prevent pulmonary embolism and postphlebitic syndrome in women during pregnancy and the puerperium. The frequency of a severe postphlebitic syndrome after our surgical approach is lower than the rates published for anticoagulation treatment alone.

No evidence for an influence of the human platelet antigen-1 polymorphism on the antiplatelet effects of glycoprotein IIb/IIIa inhibitors.

This study investigated the hypothesis that the human platelet antigen-1 (HPA-1) polymorphism may influence the antiplatelet effects of glycoprotein (GP)IIb/IIIa inhibitors. Adenosine diphosphate (30 micro mol)-induced fibrinogen binding was measured by flow cytometry. Abciximab (0.03-3 micro g/ml), tirofiban (0.3-30 nmol/l) or eptifibatide (0.01-1 micro g/ml) were incubated for 15 min with the samples prior to stimulation. IC(50) values for the inhibition of fibrinogen binding were determined from each experiment. All subjects were genotyped by GALIOS and automated fluorescence correlation spectroscopy. Although a marked variability in the inhibitory effects of all three GPIIb/IIIa inhibitors was confirmed, there were no significant differences between the genotypes with respect to the inhibition of fibrinogen binding. Thus, the present study does not provide evidence for an effect of HPA-1 polymorphism on the inter-individual variability in the platelet inhibitory effects of the three GPIIb/IIIa inhibitors approved for clinical use.

Low incidence of paradoxical platelet activation by glycoprotein IIb/IIIa inhibitors.

The human platelet antigen-1 (HPA-1, Pl(A)) polymorphism has been proposed to influence the inhibitory actions of abciximab. Thus, we hypothesized that this polymorphism might also be the cause for paradoxical activation of platelets by GPIIb/IIIa inhibitors. The effects of abciximab (1-10 microg/ml), tirofiban (3-30 nM), or eptifibatide (0.3-3 microg/ml) on basal and ADP (3 microM)-induced CD62P externalization were measured in n=62 healthy blood donors and n=177 patients with stable coronary artery disease. All subjects were genotyped for the human platelet antigen-1 (HPA-1, Pl(A)) polymorphism by GALIOS(R) and fluorescence correlation spectroscopy. Although a significant platelet hyperreactivity was observed in the patients, the HPA-1 genotype did not influence basal or ADP-induced CD62P expression. A moderate (twofold) stimulation of CD62P expression by abciximab but not by tirofiban or eptifibatide was observed in one patient. Interestingly, this patient carried the HPA-1 b/b genotype. In no other subject any activation of platelets by GP IIb/IIIa inhibitors was observed and there were no statistically significant differences between HPA-1 genotypes with respect to the effects of GP IIb/IIIa inhibitors on basal or ADP-stimulated CD62P expression. It is concluded that paradoxical platelet activation by abciximab is a rare (<2%) phenomenon. HPA-1 b/b genotype might be a contributing factor but clearly does not predict platelet activation by GP IIb/IIIa inhibitors.