Elisabetta Castoldi

Coagulation factors and the protein C system as determinants of thrombin generation in a normal population

Summary.  Background: Thrombin generation is a powerful tool to probe overall plasma coagulability.

Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera

We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2(V617F) mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2(V617F) carriers compared with noncarriers and was highest in JAK2(V617F) homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2(V617F) carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2(V617F) mutational load.

Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome

To the editor: Loss of transmembrane lipid asymmetry in apoptotic cells or in activated platelets is thought to be catalyzed by a specific membrane protein named phospholipid scramblase.[1][1] Recently, the transmembrane protein TMEM16F was shown to be required for Ca2+-induced lipid scrambling and

Regulation of coagulation by protein S.

Purpose of reviewProtein S has been one of the least mechanistically understood amongst the vitamin K-dependent coagulation proteins, and diagnosis of protein S deficiency and quantification of the associated thrombotic risk are not straightforward. In this review, the regulation of thrombin generation by protein S and the pathophysiological implications of protein S deficiency are discussed in the light of recent findings on the anticoagulant function(s) of protein S. Recent findingsProtein S expresses both activated protein C-dependent and activated protein C-independent anticoagulant activities, but the former is generally believed to be lost upon binding of protein S to C4b-binding protein. Recently it has been shown that protein S acts as a cofactor of tissue factor pathway inhibitor in the down regulation of factor X-activation, which provides a mechanistic basis for the activated protein C-independent anticoagulant activity of protein S in plasma. In addition, reevaluation of the role of the protein S/C4b-binding protein complex has demonstrated that C4b-binding protein-bound protein S does express activated protein C-cofactor activity, especially during the inactivation of factor Va Leiden. SummaryThese findings underscore the central role of protein S in the regulation of coagulation and may have important implications for the evaluation of the thrombotic risk associated with protein S deficiency.

Thrombin generation tests

The recent development of semi-automated methods has revived interest in the thrombin generation test, a global assay that measures the overall tendency of a plasma sample to form thrombin after initiation of coagulation. The thrombin generation curve, which is characterised by a lag phase followed by the formation and subsequent inhibition of thrombin, reflects all three phases of coagulation (initiation, propagation and termination). However, the specific contribution of each coagulation factor or inhibitor to the assay outcome depends on the reaction conditions used (e.g. tissue factor concentration used to trigger coagulation, addition of thrombomodulin or activated protein C). Although several studies have shown a correlation between thrombin generation and the risk of bleeding or venous thrombosis, the application of thrombin generation assays to clinical decision-making is still hampered by standardisation problems. The present paper discusses these issues with particular reference to Calibrated Automated Thrombography.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca(2+)-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency

Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 +/- 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 +/- 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 +/- 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding.

APC resistance: biological basis and acquired influences

Summary.  Proteolytic inactivation of factors Va (FVa) and VIIIa (FVIIIa) by activated protein C (APC) and its cofactors protein S and factor V (FV) is a key process in the physiological down‐regulation of blood coagulation. Functional abnormalities of this pathway, which manifest themselves in vitro as a poor anticoagulant response of plasma to added APC (APC resistance), are prevalent in the general population and are associated with an increased risk of venous thrombosis. APC resistance was originally discovered in thrombophilic families and later shown to be associated with the common FV Arg506Gln (FVLeiden) mutation, which abolishes one of the APC‐cleavage sites in FV. Although FVLeiden is the major cause of hereditary APC resistance, it is becoming increasingly clear that several other genetic and acquired conditions contribute to APC resistance and thereby increase the risk of venous thrombosis. This paper reviews the multifactorial etiology of APC resistance and discusses its clinical implications.

Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma

Summary.  Background: Protein S and tissue factor pathway inhibitor (TFPI) act together in down‐regulating coagulation. Objective: To investigate the TFPI/protein S system in hereditary and acquired protein S deficiency. Methods: Plasma antigen levels of protein S and full‐length TFPI were determined in heterozygous type I protein S‐deficient individuals (n = 35), patients on oral anticoagulant treatment (OAT) (n = 29), oral contraceptive (OC) users (n = 10) and matched controls. Thrombin generation was determined using calibrated automated thrombography. Results: Full‐length TFPI levels were lower in type I protein S‐deficient individuals (76.8 ± 33.8%) than in age‐ and sex‐matched controls (128.0 ± 59.4%, P < 0.001). Among protein S‐deficient individuals with thrombosis, those on OAT had not only lower total protein S levels (25.7 ± 8.2% vs. 54.7 ± 8.2%, P < 0.001), but also lower full‐length TFPI levels (52.6 ± 15.0% vs. 75.4 ± 22.9%, P = 0.009) than those not on OAT. Similarly, OC users had lower protein S (73.8 ± 11.5% vs. 87.9 ± 10.8%, P = 0.005) and full‐length TFPI levels (73.7 ± 27.7% vs. 106.4 ± 29.2%, P = 0.007) than non‐users. When triggered with tissue factor, plasma from protein S‐deficient individuals generated 3–5‐fold more thrombin than control plasma. The difference was only partially corrected by normalization of the protein S level, full correction requiring additional normalization of the TFPI level. Protein S‐immunodepletion experiments indicated that free protein S and full‐length TFPI form a complex in plasma, and the protein S/TFPI interaction was confirmed by surface plasmon resonance analysis. Conclusions: Full‐length TFPI binds to protein S in plasma and is reduced in genetic and acquired protein S deficiency. The concomitant TFPI deficiency substantially contributes to the hypercoagulable state associated with protein S deficiency.

Advances in understanding the bleeding diathesis in factor V deficiency

Coagulation factor V (FV), present in plasma and platelets, is an indispensable clotting factor, as demonstrated by the uniform lethality of FV knock‐out mice. Surprisingly, however, severe FV deficiency is rarely fatal in humans. In fact, although several cases of life‐threatening intracranial haemorrhage have been reported in FV‐deficient newborns, many patients with undetectable FV levels experience only mild to moderate bleeding and do not require routine prophylaxis. While the reasons for this variable phenotypic expression are largely unknown, several observations from different laboratories indicate platelets as crucial players in FV deficiency. Moreover, we have recently shown that plasma levels of tissue factor pathway inhibitor are considerably reduced in FV‐deficient plasma, which results in enhanced thrombin generation especially at very low FV levels (<2%). The present review discusses and integrates these findings in the context of the biology of FV and the clinical features of FV deficiency.