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The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl.The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype.Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2.Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (tThe p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Current guidelines recommend caution in prescribing concomitant use of direct-acting oral anticoagulants (DOACs) and antiepileptic drugs due to drug-drug interactions leading to potential risk of DOACs subtherapeutic concentration and treatment failure. Herein we report a significant interaction between carbamazepine (CZP) and apixaban, causing subtherapeutic concentration of the drug in a patient with atrial fibrillation who had a transient ischemic attack (TIA) episode. Another anti-Xa DOAC, edoxaban, administered to the patient after TIA occurrence did not show significant interaction with CZP. In addition to confirm that cautions should be used when antiepileptic and DOACs are concomitantly prescribed, the present case also demonstrates that, in the management of certain subsets of patients who need anticoagulant treatment, measurement of DOAC plasma concentration can help guide a personalized management and avoid adverse clinical outcomes.

Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alterations have been described in novel coronavirus pneumonia patients, case-control studies of von Willebrand factor (VWF), factor VIII (FVIII), and a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) are lacking. VWF, ADAMTS13, FVIII, D-dimer, C-reactive protein, and routine blood cells and chemistry were measured in 10 novel coronavirus pneumonia patients and 10 non-novel coronavirus pneumonia controls. Hemostatic factors were measured less than 48 h of hospital admission in patients without invasive ventilation. D-Dimer, C-reactive protein, and fibrinogen concentrations, high in both groups, did not differ significantly in novel coronavirus pneumonia vs. non-novel coronavirus pneumonia patients. Median VWF-antigen (324 vs. 153 IU/dl, P 

Background Acquired von Willebrand factor (VWF) deficiency was described in Philadelphia‐negative myeloproliferative neoplasms, especially in essential thrombocythemia (ET). VWF phenotype in contemporary patients with polycythemia vera (PV) remains less explored. Objectives To characterize the VWF phenotype in PV and to compare VWF phenotype in PV with matched healthy subjects and ET patients. Patients/Methods We studied 48 PV patients, treated according to current recommendations (hematocrit ≤ 45%, on low‐dose aspirin prophylaxis); 48 healthy and 41 subjects with ET, all sex, age, and blood group matched. We measured VWF antigen, activity, multimeric pattern, ADAMTS‐13, and factor VIII (FVIII) antigen. Results In patients with PV, VWF antigen and activity were significantly higher than in healthy subjects (antigen: 119[96‐137] vs 93[79‐107] IU/dL; activity: 114[95‐128] vs 90[79‐107] IU/dL, respectively, medians and interquartile, P

We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D′D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient’s daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B–like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

The direct oral anticoagulant apixaban (APX), a strong factor Xa inhibitor, binds also to plasma proteins, especially albumin, and minimally to α1-acid glycoprotein. Although APX can cross the red cell membrane, due to its chemical structure, and could bind to haemoglobin (Hb), no investigation was performed on this possible phenomenon that could affect the APX plasma concentration and thus its pharmacokinetics and pharmacodynamics. We addressed this issue by (1) measuring the levels of APX and haematological/biochemical parameters in 90 patients on APX therapy; (2) assessing the effect of APX on oxygen saturation curves of Hb; (3) testing the direct APX binding to Hb by fluorescence spectroscopy and a zinc-induced precipitation of Hb coupled to a reversed-phase high-performance liquid chromatography (HPLC)-based method; and (4) simulating in silico by molecular docking the APX interaction with human Hb. In a multivariable analysis, Hb was the only independent variable significantly and inversely associated in 90 patients with APX peak plasma level, at variance with patients treated with rivaroxaban (n = 86) and dabigatran (n = 34) therapy. APX causes a progressive left-shift of the oxygen dissociation curve of purified Hb solution, with a Kd ≅300 µM. Fluorescence- and HPLC-based assays concordantly showed that APX binds to Hb with a Kd ≅350 µM. Finally, docking simulations showed that APX can fit into in the central cavity of Hb. These findings support the hypothesis that APX does bind to Hb, which, due to its millimolar concentration in blood, can act as ‘buffer’ for the drug and consequently affect its free plasma level.

Summary Background Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. Objectives We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. Patients/Methods Sixty-nine ET patients (M = 29; median age, 62 [48–70] years; platelets, 432 [337–620] × 103 μL−1), 69 matched controls and 10 subjects with reactive thrombocytosis (RT) were studied. VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoproteinV (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. Results In ET patients, VWF:Ag was increased by 31 ± 13% vs. controls (P

Background : Double filtration plasmapheresis (DFPP) is a form of therapeutic plasma exchange (TPE) that removes high-molecular-weight (HMW) pathological mediators from the plasma with two filters: a plasma separator and a plasma fractionator. Relative to simple TPE, the semi-selectiveness of DFPP reduces protein loss and the need for substitution fluid. However, depending on the choice of plasma fractionator, DFPP may negatively impact hemostatic components such as HMW coagulation factors. Aim: To determine the impact of DFPP on various hemostatic parameters (i.e. FVIII activity, fibrinogen level, vWF level and activity, ADAMTS13 level and activity, protein C (PC) activity, protein S (PS) activity, antithrombin (AT) activity, prothrombin time (PT), and activated partial thromboplastin time (aPTT)). Methods: Fourteen patients undergoing weekly, bimonthly, or monthly DFPP sessions for hematologic conditions (n=11) or nervous system disorders (n=3) were recruited. Hemostatic parameters were measured immediately before and after 27 DFPP sessions (1-4 sessions/patient). The treatment volume was standardized at one plasma volume, and anticoagulation was performed with ACD-A citrate dextrose solution. EC-30W and EC-50W were used as the plasma fractionators in 4 and 23 sessions, respectively. In addition to primary data collection, we systematically searched PubMed, MEDLINE, and EMBASE for studies that investigated the impact of DFPP on hemostasis. No restriction was placed on filter choice. Results: In our cohort of 14 patients, all hemostatic changes were statistically significant. After a routine DFPP session, the level and/or activity of HMW proteins (>100kDa: FVIII, fibrinogen, vWF, ADAMTS13) were decreased more than those of low-molecular-weight (LMW) proteins ( Our systematic review included 26 cohort studies, 6 case reports, and 1 randomized controlled trial. Increases in INR and aPTT following DFPP were considerably higher in previous studies, which may relate to the commonality of heparin-induced anticoagulation. The present study is the first comprehensive investigation of the impact of DFPP on hemostatic parameters with such a large sample size. It is also the first study of its nature to measure ADAMTS13-related parameters. Conclusions: Routine DFPP resulted in significant reduction across all investigated hemostatic proteins and significant prolongation of clotting tests. The activities of HMW coagulation-related proteins were decreased more than those of LMW anticoagulation-related proteins. This finding suggests that DFPP may increase overall bleeding risk, a consideration for patient safety that may be of importance in continuous DFPP treatment. Additional high quality evidence is needed to elucidate the effect of DFPP on the hemostatic system. Disclosures Matino: Sanofi: Honoraria; Sobi: Honoraria, Research Funding; Roche: Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bioviiix: Honoraria.

SummaryCongenital thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy, inherited with autosomal recessive mode as a dysfunction or severe deficiency of ADAMTS-13 (A Disinte-grin And Metalloprotease with ThromboSpondin 1 repeats Nr. 13), caused by mutations in the ADAMTS-13 gene. About 100 mutations of the ADAMTS-13 gene were identified so far, although only a few char-acterised by in vitro expression studies. A new Asp to Gly homozygous mutation at position 173 of ADAMTS-13 sequence was identified in a family of Romanian origin, with some members affected by clinical signs of TTP. In two male sons, this mutation caused a severe (< 3 %) deficiency of ADAMTS-13 activity and antigen level, associated with periodic thrombocytopenia, haemolytic anaemia and mild mental confusion. Both parents, who are cousins, showed the same mutation in heterozygous form. Expression studies of the mutant ADAMTS-13, performed in HEK293 cells, showed a severe decrease of the enzyme’s activity and secretion, although the protease was detected inside the cells. Molecular dynamics found that in the D173G mutant the interface area between the metalloprotease domain and the disintegrin-like domain significantly decreases during the simulations, while the proline-rich 20 residues linker region (LR, 285–304) between them undergoes extensive conformational changes. Inter-domain contacts are also significantly less conserved in the mutant compared to the wild-type. Both a decrease of the inter-domain contacts along with a substantial conformational rearrangement of LR interfere with the proper maturation and folding of the mutant ADAMTS-13, thus impairing its secretion.Supplementary Material to this article is available online at www.thrombosis-online.com.

Prothrombin deficiency is among the rarest inherited coagulation disorders, with a prevalence of approximately 1:2,000,000. Two main phenotypes can be distinguished: (1) hypoprothrombinemia (type I deficiency), characterized by concomitantly low levels of activity and antigen; and (2) dysprothrombinemia (type II deficiency), characterized by the normal or near-normal synthesis of a dysfunctional protein. In some cases, hypoprothrombinemia associated with dysprothrombinemia was also described in compound heterozygous defects. No living patient with undetectable plasma prothrombin has been reported to date. Prothrombin is encoded by a gene of approximately 21 kb located on chromosome 11 and containing 14 exons. Forty different mutations have been identified and characterized in prothrombin deficiency. Many of them surround the catalytic site, whereas another "hot spot" is localized in the recognition domain called anion binding exosite I, also called fibrinogen recognition site. Recently, mutations were identified also in the Na (+)-binding loop and in the light A-chain of thrombin. Most hypoprothrombinemia-associated mutations are missense, but there are also nonsense mutations leading to stop codons and one single nucleotide deletion. Finally, the main aspects of clinical manifestations and therapy of congenital prothrombin deficiency are presented and discussed.

Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor.The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0-60 dyn cm(-2) range.WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL(-1) already bound to the receptor. By contrast, the WT and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GPIbα (Kd ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of 14 dyn cm(-2) , the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of 30 dyn cm(-2) . Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein.The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites.

Prothrombin (factor II [FII]) deficiency is a rare inherited coagulation disorder, having a prevalence of approximately 1 in 2,000,000. Two phenotypes can be distinguished: (1) true hypoprothrombinemia (type I deficiency), characterized by concomitantly low levels of the zymogen antigen; and (2) dysprothrombinemia (type II deficiency), characterized by the normal or near-normal synthesis of a dysfunctional protein. In the latter case, recent studies showed that particular mutations in the catalytic domain of active thrombin can even impair the enzyme interaction with antithrombin, favoring thromboembolic diseases. In some cases, hypoprothrombinemia associated with dysprothrombinemia was also described in compound heterozygous defects. Prothrombin is essential for the development of mammalian organisms. No living patient with undetectable plasma prothrombin has been reported to date. Prothrombin is encoded by a ≈21 kb gene located on chromosome 11 and containing 14 exons. Thirty-nine different mutations have been identified and characterized in prothrombin deficiency. Many of these are present in the catalytic site, whereas some involve regulatory domains, such as the anion-binding exosite I, the Na+-binding loop, and the light A-chain. Most hypoprothrombinemia-associated mutations are missense, but nonsense mutations leading to stop codons and one single nucleotide deletion have also been identified. Finally, recent developments in the therapy of congenital prothrombin deficiency are presented and discussed.

The thrombogenic activity of Von Willebrand factor (VWF) is proportional to its molecular size and inversely related to its proteolysis by ADAMTS-13. Oxidation of VWF severely impairs its proteolysis by the metalloprotease. This study was aimed at assessing in patients with type 1 and type 2 diabetes whether protein carbonyls, marker of oxidative stress, are associated with both the level and oxidation status of VWF as well as with micro- and macroangiopathic complications. Eighty-three diabetic patients (41 type 1 and 42 type 2 diabetic subjects) and their respective eighty-three healthy controls were studied after verifying the availability, through institutional databases, of clinical biochemistry records spanning at least 3 years. VWF and protein carbonyls were measured by immunoassays, whereas VWF multimers were studied by SDS-agarose gel electrophoresis. Type 2 diabetic subjects had higher levels of VWF antigen (VWF:ag), VWF activity (VWF:act) and plasma proteins’ carbonyls compared to both their controls and type 1 diabetic subjects. Moreover, high molecular weight VWF multimers and specific VWF-bound carbonyls were significantly increased in subjects with micro- and macro-angiopathic complications. In both type 1 and type 2 diabetic subjects, ADAMTS-13 activity was in the normal range. In a multivariable analysis, only VWF-bound carbonyls were significantly associated with any form of thrombotic angiopathy in the entire group of T1- and T2 diabetic patients. These data provide first evidence that not only high VWF levels but also its oxidation status and the presence of high molecular weight VWF multimers that are not observed in SDS-agarose gel electrophoresis of normal subjects are associated with thrombotic angiopathies in diabetes mellitus. These findings may help identify diabetic patients particularly at risk for these complications and elucidate a new pathophysiological mechanism of thrombotic angiopathies in this clinical setting.

Summary. Background: Binding of von Willebrand factor (VWF) multimers of ultra-large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). Objective: To assess the potential of VWF-related measurements as markers of disease activity and severity in TTP. Methods: VWF antigen (VWF:Ag), platelet glycoprotein-Ib-α binding-conformation (GPIb-α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co-factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. Results: VWF:Ag and VWF-GPIb-α/BC were higher in TTP patients than controls (P

Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded “cysteine knot” domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).

ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A D isintegrin A nd M etalloprotease with T hrombo S pondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain and 2 CUB ( C omplement c1r/c1s, sea U rchin epidermal growth factor, and B one morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr 1605 -Met 1606 ) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this review, we a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b) address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c) indicate the direction of future investigations

Abstract 3306 Background. Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods. In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results. The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values Conclusions. These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb. Disclosures: No relevant conflicts of interest to declare.

The product of thrombin digestion of fibrinogen, that is fibrin, binds to thrombin with high specificity. The latter interacts with two classes of binding sites on fibrin, one of low affinity in the E domain and the other of high affinity in the D domain of fibrin(ogen) molecules. Binding of thrombin to fibrinogen involves sequences of both Aα and Bβ chain in the fibrinogen E domain. This recognition sites are still able to interact with thrombin after cleavage of fibrinopeptide A and B and form the low affinity binding site for the enzyme. The D domains contain a γ chain variant, termed γ′, arising from an alternative mRNA splicing, resulting in an elongated chain composed of 427 instead of 411 residues. The inserted region at the C-terminus is composed of 20 amino acids (408VRPEHPAETEYDSLYPEDDL427), rich of acidic residues. The γ′ chain, mainly heterodimerizes in the fibrinogen molecule with the more abundant γA chain, thus generating the γA/γ′ dimers (about 10% of the total γ chain content). The different expression of γ′ chain has been variably associated with both venous and arterial thrombosis. Previous genetic studies showed that the fibrinogen γ-H2 haplotype is characterized by a reduced fibrinogen γ′ levels. This haplotype is associated with a significantly increased risk for venous thrombosis. At variance with venous thromboembolism, the effect of altered expression of γ′ chain on arterial thrombosis remains largely elusive and still debated. Biochemical studies showed that γ′ chains bind to α-thrombin with high affinity and that the 408-427 region of γ′ chain binds to the anion binding exosite (ABE)-II of thrombin. We can speculate that the fibrinogen γ′ chain, through its ability to bind to thrombin, might enhance the amount of clot-bound thrombin. The latter may represent a storage pool of the enzyme, facilitating arterial thrombus formation via platelet activation. In this study, we investigated the effect of the fibrinogen γ′ and of its 20-amino acid-insertion peptide on the thrombin interaction with the platelet receptors glycoprotein (Gp) Ibα, and protease-activated receptors PAR-1 and PAR-4, involved in platelet activation. Fragment D was used as the best surrogate to selectively study the high affinity binding site for thrombin in γ chain in a conformation similar to that present in the native fibrinogen molecule. Both synthetic γ′ peptide and fragment D*, containing the elongated γ′ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC50 values of 42±3.5 μM and 0.47±0.03 μM, respectively. Solid phase binding and titration spectrofluorimetric assays analyzing the tryptophan fluorescence of thrombin showed that both fragment D* and the synthetic γ′ peptide specifically bind to ABE-II of thrombin and competitively inhibit the thrombin interaction with GpIbα with a mean Ki≈0.5 μM and ≈35 μM, respectively. HPLC assays showed that both the γ′ chain allosterically inhibited thrombin cleavage of the synthetic PAR-1 38-60 peptide, with an IC50 value of 45 μM, in good agreement with the Kd value of thrombin binding to γ′ chain derived from fluorimetric titrations. Likewise, flow cytometry studies showed that the hydrolysis of native PAR-1 molecules on intact platelets was progressively inhibited by increasing concentrations of both fragment D* and γ′ peptide, while PAR-4 cleavage was unaffected. In summary, fibrinogen γ′ chain binds with high affinity to thrombin and inhibits with combined mechanisms the thrombin-induced platelet activation. The role of different expression of γ′ chain in circulating fibrinogen may variably influence the thrombotic and hemorrhagic manifestations in different clinical settings or different phases of thrombotic diseases.