Annual incidence and severity of acute episodes in hereditary thrombotic thrombocytopenic purpura.

Hereditary thrombotic thrombocytopenic purpura (hTTP) is a rare thrombotic microangiopathy characterized by severe congenital ADAMTS13 deficiency and recurring acute episodes causing morbidity and premature death. Information on the annual incidence and severity of acute episodes in patients with hTTP is largely lacking. This study reports prospective data on 87 patients from the Hereditary TTP Registry (clinicaltrials.gov #NCT01257269) for survival, frequency, and severity of acute episodes from enrollment until December 2019. The 87 patients, followed up for a median of 4.2 years (range, 0.01-15 years), had a median age at overt disease onset and at clinical diagnosis of 4.6 years and 18 years (range, 0.0-70 years for both), respectively. Forty-three patients received regular plasma prophylaxis, whereas 22 did not, and treatment changed over time or was unknown in the remaining 22. Forty-three patients experienced 131 acute episodes, of which 91 (69%) occurred in patients receiving regular prophylaxis. This resulted in an annual incidence of acute episodes of 0.36 (95% confidence interval [CI], 0.29-0.44) with regular plasma treatment and of 0.41 (95% CI, 0.30-0.56) without regular plasma treatment. More than one-third of acute episodes (n = 51) were documented in children <10 years of age at enrollment and were often triggered by infections. Their annual incidence of acute episodes was significantly higher than in patients aged >40 years (1.18 [95% CI, 0.88-1.55] vs 0.14 [95% CI, 0.08-0.23]). The prophylactic plasma infusion regimens used were insufficient to prevent acute episodes in many patients. Such regimens are burdensome, and caregivers, patients, and their guardians are reluctant to start regular plasma infusions, from which children particularly would benefit.

The Homozygous Type II Antithrombin Deficient Pregnant Woman Monitored by Thrombin Generation Assay.

BACKGROUND: We present the case study of a 28-year-old pregnant woman with antithrombin deficiency who was treated with low-molecular-weight heparin (LMWH). METHODS: Due to severe homozygous type II antithrombin heparin binding site (HBS) deficiency, the thrombin generation (TG) was monitored in this woman via the Thrombin Generation Assay (TGA). We used Siemens diagnostic kits Berichrom® Antithrombin III (IIa) and INNOVANCE® Antithrombin (Xa) to determine antithrombin activity. We used a chromogenic method for determination of factor Xa (FXa) inhibition. RESULTS: There were no thrombotic complications during the whole pregnancy of the observed woman. Antithrombin was administered before and after delivery, which was significantly reflected in the decrease in thrombin generation. CONCLUSIONS: Consistent monitoring of thrombin generation with LMWH anticoagulant therapy administration during pregnancy together with antithrombin administration before and after delivery can improve the overall condition of pregnant women and the quality of their care.

Extracorporeal membrane oxygenation seems to induce impairment of primary hemostasis pathology as measured by a Multiplate analyzer: An observational retrospective study.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) support is often associated with bleeding complications caused by secondary or primary hemostasis pathology. However, there are limited data investigating primary hemostasis using Multiplate aggregometry with specific diagnostics tests for vWF (von Willebrand factor) deficiency. AIMS: The aim of this study was to find out whether short-term ECMO produces the pathology of primary hemostasis that is detected by Multiplate aggregometry and to investigate the pathology of vWF. METHODS: In this study, blood samples of 20 patients undergoing lung transplantations with short-term perioperative ECMO support were analyzed. The multimeric structure, the levels of von Willebrand factor antigen (vWF), ristocetin cofactor (RCo), collagen-binding protein (CB), and the results of multiple electrode aggregometry RISTO (ristocetin), ADP (adenosine diphosphate), ASPI (Aspirin®; arachidonic acid), and TRAP (thrombin receptor activating peptide) tests were compared to the samples obtained before and after ECMO support. RESULTS: The Multiplate ADP and RISTO tests showed the presence of significant pathology in primary hemostasis after surgery (p < 0.05), suggesting the presence of acquired platelet dysfunction. Although the RISTO tests suggest the presence of acquired vWF deficiency, laboratory tests for vWF antigen and RCo and CB tests showed an increase in this case. The multimeric structure of vWF did not show clinically significant deterioration. CONCLUSIONS: Multiple aggregometry ADP, ASPI, and TRAP tests seem to be able to detect primary hemostasis pathology (platelets aggregation and adhesion pathology) that is present during short-term perioperative ECMO support in lung transplantation procedures. Interestingly, RISTO tests seem to be more suitable for the diagnosis of platelet dysfunction than the diagnosis of acquired vWF deficiency in this situation.

Diagnosing Czech Patients with Inherited Platelet Disorders.

A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.

Hereditary antithrombin deficiency in pregnancy - severe thrombophilic disorder as a danger for mother and foetus

SETTING: In the article, we remember the role of antithrombin (AT) in hemostasis, escalation of AT-potential with heparin and difficulties with monitoring the effectiveness of LMWH therapy (low molecular weight heparin) in patients with AT deficiency. We pay most of our attention to hereditary AT deficiency and its thromboembolic risk in pregnancy. METHODS: In the introduction, the principle of AT function, its two main domains and the regulation of synthesis are cleared. We describe the causal mutations of hereditary AT deficiency in SERPINC1 gen and the relation to a thromboembolic risk. The general recommendations for patients with hereditary AT deficiency and pregnant women are mentioned. As the risk of thromboembolic disease is escalated in pregnancy, the LMWH should always be considered. There has been frequently observed that patients with AT deficiency do not elevate anti-Xa-levels when standard prophylactic LMWH doses are used. This fact well illustrates that heparin without AT may not inhibit the active coagulant factors efficiently enough. Therefore, if a high thromboembolic risk in the patient's anamnesis is present, the LMWH dosing should be escalated. In individual cases, concomitant administration of an antithrombin concentrate to the heparin treatment is recommended at the time of delivery or in the case of deep venous thrombosis. In this article, three cases of unusual pregnancy in patients with different types of AT deficiency are reported. The case reports are summarized from the Department of Hematology at Hospital Kolín, the Centre of Hemostasis and Thrombosis at Institute of Hematology and Blood Transfusion in Prague and from cooperating obstetrical departments in the Czech Republic. RESULTS: We demonstrated the threat of hereditary AT deficiency in three case reports. In one case, the estimated risk of thromboembolism ­ type I of AT-deficiency (quantitative) ­ was in a good correlation with real peripartal complications. In the next two cases with different types of AT deficiency, we showed surprising courses of complicated pregnancies. CONCLUSION: As it has been shown, it is not safe to estimate the risk of thromboembolism on the base of causal mutation for AT deficiency. For present clinical practice, we should still remember AT deficiency as a potentially very dangerous thromboembolic disorder for mother and fetus; thus, excellent cooperation of an obstetrician and a hematologist is necessary.

Identifying risk factors and optimizing standard of care for patients with acquired haemophilia A: Results from a Czech patient cohort.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare autoimmune disorder, characterized by bleeds of varying severity caused by autoantibodies against factor VIII (FVIII). AIM: Identify risk factors associated with AHA-related deaths/relapses and assess the effect of increased corticosteroid doses. METHODS: AHA patients treated across two specialist centres in the Czech Republic, generally receiving first-line haemostatic therapy with rFVIIa and immunosuppression with corticosteroids/cyclophosphamide, were included. We analysed the association between early death (within 8 weeks of diagnosis [considered disease-related]) and age, malignancy, FVIII levels and bleeding severity. Risk factors associated with reduced 2-year survival and relapse incidence, and the effect of increased corticosteroid doses on early death and remission were also assessed. RESULTS: The demographics of the described cohort (n = 66) were similar to other AHA registries. Early death occurred in 20% of cases. Unlike age and malignancy, FVIII levels <1% and severe bleeding were associated significantly with early death (P = .010 and P = .046, respectively). Patients with underlying malignancy or requiring continued haemostatic therapy exhibited significantly decreased 2-year survival compared with those without these risk factors (P = .007 and P = .006, respectively). Patients with an underlying autoimmune disease relapsed significantly more than those without (P = .015). Higher corticosteroid doses were associated with a significantly increased incidence of early deaths (P < .001), but also with early remission (P < .001). CONCLUSION: Based on this rather large patient cohort, we were able to evaluate the significance of several risk factors associated with treatment outcomes in AHA and the effect of initial treatment with corticosteroids on survival and time to remission.

Molecular basis and outcomes of atypical haemolytic uraemic syndrome in Czech children.

Atypical haemolytic uraemic syndrome is an ultra-rare, life-threatening disease. Causative variants in genes that encode complement factors can be identified in 40-70% of cases. We performed genetic analysis of 21 Czech children with atypical haemolytic uraemic syndrome. Genetic or acquired predisposition to the disease was identified in the majority of our patients: CFHR1 and CFHR3 deletions in 14/21 (67%; 13 of them were positive for anti-complement factor H antibodies), variants in complement genes or DGKE in 13/21 (62%). Multiple genetic findings were identified in eight patients (38%). The incidence of atypical haemolytic uraemic syndrome in the Czech paediatric population was estimated to be 0.092 (CI 0.053-0.131) cases per million inhabitants and 0.92 (CI 0.53-1.32) cases per 100,000 births for the entire reporting period. Ten patients were initially treated with plasma exchange and eight with eculizumab or with a combination of eculizumab and plasma exchange. At the last follow-up, 20 patients were alive and one patient had end-stage renal disease.Conclusion: The incidence of atypical haemolytic uraemic syndrome in the Czech paediatric population corresponds to the reported incidence in Europe. We detected the unusually high rate of CFHR1/CFHR3 deletions associated with anti-complement factor H antibodies in Czech paediatric patients. Treatment by eculizumab led to superior outcomes and prevention of the disease relapses compared with plasma exchange therapy. Our results may help to understand the polygenic nature of atypical haemolytic uraemic syndrome as a disease that results from a combination of various risk factors. What is Known: • Atypical haemolytic uraemic syndrome (aHUS) is considered a polygenic and multifactorial disease. Genetic predisposition to aHUS is identified in 40-70% of children. • Anti-complement factor H antibodies are usually found in 6-25% of affected children. What is New: • Potentially causative genetic or acquired factors were confirmed in the majority of patients. The prevailing finding was the unusually high rate of CFHR1/CFHR3 deletions associated with anti-complement factor H antibodies (62% of patients). • The incidence of aHUS in Czech children is 0.092 (CI 0.053-0.131) cases per million inhabitants and 0.92 (CI 0.53-1.32) cases per 100,000 births for the entire reporting period.

The International Hereditary Thrombotic Thrombocytopenic Purpura Registry: key findings at enrollment until 2017.

Congenital thrombotic thrombocytopenic purpura is an autosomal recessive inherited disease with a clinically heterogeneous course and an incompletely understood genotype-phenotype correlation. In 2006, the Hereditary TTP Registry started recruitment for a study which aimed to improve the understanding of this ultra-rare disease. The objective of this study is to present characteristics of the cohort until the end of 2017 and to explore the relationship between overt disease onset and ADAMTS13 activity with emphasis on the recurring ADAMTS13 c.4143_4144dupA mutation. Diagnosis of congenital thrombotic thrombocytopenic purpura was confirmed by severely deficient ADAMTS13 activity (≤10% of normal) in the absence of a functional inhibitor and the presence of ADAMTS13 mutations on both alleles. By the end of 2017, 123 confirmed patients had been enrolled from Europe (n=55), Asia (n=52, 90% from Japan), the Americas (n=14), and Africa (n=2). First recognized disease manifestation occurred from around birth up to the age of 70 years. Of the 98 different ADAMTS13 mutations detected, c.4143_4144dupA (exon 29; p.Glu1382Argfs*6) was the most frequent mutation, present on 60 of 246 alleles. We found a larger proportion of compound heterozygous than homozygous carriers of ADAMTS13 c.4143_4144dupA with overt disease onset at < 3 months of age (50% vs 37%), despite the fact that ADAMTS13 activity was <1% in 18 of 20 homozygous, but in only 8 of 14 compound heterozygous carriers. An evaluation of overt disease onset in all patients with an available sensitive ADAMTS13 activity assay (n=97) shows that residual ADAMTS13 activity is not the only determinant of age at first disease manifestation. Registered at clinicaltrials.gov identifier NCT01257269.

Diagnostic strategies in disorders of hemostasis.

Hemostasis is a complicated biological system, where the balance between procoagulation and anticoagulation processes maintains fluidity of blood through intact blood vessels and creates thrombi when it is needed to prevent bleeding from the impaired vessels. The modern model of hemostasis is divided into 2 principal phases, the first being defined as primary hemostasis which involves the platelet-vessel interplay, while the second, defined as secondary hemostasis, mainly involves coagulation factors and surfaces of activated cells. The activation and amplification of the coagulation cascade is regulated by natural inhibitors of coagulation. The blood clots which arise to prevent loss of blood must subsequently be broken down and the compact blood vessel wall must be restored. This process is called fibrinolysis. Bleeding and thromboses are manifestations of the impaired hemostatic balance. Detection of its cause is important for efficient treatment and prevention of the condition. This requires a combined evaluation of the family and personal history, a clinical anamnesis along with the evaluation of laboratory results. It is not reasonable or economical to perform all the available tests of hemostasis at the same time. It is recommendable to proceed from the global to the screening and then special tests and, through the process of elimination, obtain an explanation of the bleeding or prothrombotic phenotypes. The purpose of this report is to provide a brief overview of the principal causes of the hemostatic disorders and the practices which facilitate their diagnosis. Key words: diagnosis of hemorrhagic manifestations - diagnostics of thrombophilias - DIC diagnostics - hemophilia A - thrombopathy - von Willebrand disease.

ECMO produces very rapid changes in primary hemostasis detected by PFA-200 during lung transplantation: An observational study.

BACKGROUND: The pathology of primary hemostasis is a common complication of extracorporeal membrane oxygenation (ECMO) support. Scientific data describing its changes in patients on short-term ECMO support and the ability and speed of the restoration of its functions are limited. AIMS: The aim of this study was to describe the pathology of primary hemostasis induced by short-term ECMO support and its development over time using PFA-200, ROTEM platelet, and von Willebrand factor (vWF) analyses. METHODS: In patients undergoing lung transplantation surgery using intraoperative veno-arterial ECMO support, blood samples were analyzed using the following tests: PFA-200, ROTEM platelet tests, vWF antigen, ristocetin cofactor (RCo), and collagen-binding protein (CB) before, during, and after ECMO support. RESULTS: Blood samples from 32 patients were analyzed. All 3 PFA-200 tests (COL/EPI, COL/ADP, and COL/P2Y) showed significant deterioration during ECMO support with rapid restoration after ECMO cessation (p < 0.05), suggesting an ECMO-induced primary hemostasis disorder. A significant increase of vWF antigen after ECMO cessation (p < 0.05) was found with an increase of RCo and CB levels, although it was not significant (p > 0.05). CONCLUSIONS: Short-term ECMO support induces primary hemostasis pathology. It occurs immediately after initiation but is rapidly restored after ECMO cessation, which is detectable by PFA-200. Despite there being persistent platelet dysfunction after ECMO cessation, as seen with the ROTEM platelet results, the increased levels of vWF antigen might explain the normal results of primary hemostasis detected by PFA-200.

Effects of frozen storage conditions and freezing rate on the stability of coagulation proteins in human plasma.

OBJECTIVES: Degradation of coagulation proteins in frozen plasma may influence assay results. The aims of this study were to explore the changes in coagulation parameters in patient plasma and internal quality control (IQC) after different freezing and storage conditions during the short-term and long-term periods. METHODS: Platelet poor plasma was prepared from citrated peripheral blood collected from a group of healthy donors. The plasma was pooled, frozen and stored in a variety of freezing and storage conditions. The changes were monitored using routine coagulation assays, as well as factor VIII (FVIII) and protein S (PS) assays. RESULTS: Plasma stored in liquid nitrogen (LN 2 ) or in -80°C showed long-term stable values for routine tests for a period of over 12 months, and 6 months for FVIII. Interestingly, the activated partial thromboplastin time (aPTT) showed a temporary significant prolongation over the first two weeks. Plasma frozen and stored in -40°C is not viable for aPTT and FVIII testing, otherwise it can be used for other parameters for up to 4 months. PS showed a significant increase in all frozen samples. Freezing rate has a significant impact on plasma quality and the final storage temperature influences the long-term stability. CONCLUSION: The optimal storage conditions are ultra-low temperatures (LN 2 or -80°C) and the highest freezing rate possible. However, frozen plasma is not viable for IQC of aPTT during a period of two weeks after freezing. This study is unique in its conception as a practical guide for the handling of frozen plasma samples in modern laboratory settings.

Thrombin Generation Decrease After LMWH Administration in an Antithrombin-Deficient Pregnant Woman With a Homozygous HBS II Mutation.

The cases of antithrombin (AT)-deficient pregnant women with a homozygous HBS II mutation are relatively rare and are accompanied by an increased thrombophilic risk, which is manifested by increased thrombin generation (TG). It is very difficult to ensure their prophylactic treatment during pregnancy. We aimed to determine the utility of the thrombin generation assay (TGA) and anti-factor Xa (anti-FXa) test to monitor the effects of a prophylactic dose of low-molecular-weight heparin (LMWH) in a 28-year-old woman with homozygous AT deficiency caused by mutation c.391C > T#, (p.Leu131Phe†) in the SERPINC1 gene and to compare the findings with those from a group of pregnant and non-pregnant women also treated with LMWH. TG monitoring was chosen due to severe AT deficiency that was manifested by low levels of anti-FXa activity when monitoring the efficacy of LMWH treatment. A significant decrease in TG was detected in all monitored groups (P < .05). There were no thrombotic complications during the whole pregnancy of the woman with AT deficiency. Consistent monitoring of TG with LMWH anticoagulant therapy administration during pregnancy together with AT administration before and after delivery may improve the overall condition of pregnant women and the quality of their care.

The impact of PROS1 mutation position on thrombotic risk in protein S-deficient patients.

Background: Inherited protein S deficiency is a thrombophilic risk factor associated with venous thromboembolism. However, there is not much data on the impact of mutation position on thrombotic risk. Objectives: The aim of this study was to evaluate the risk of thrombosis due to mutations located in the sex hormone-binding globulin (SHBG)-like region as opposed to the rest of the protein. Methods: Genetic analysis of PROS1 was performed in 76 patients with suspected inherited protein S deficiency, and the effect of missense mutations present in the SHBG region on thrombosis risk was analyzed by statistical methods. Results: We found 30 unique mutations (13 of them novel), of which 17 were missense mutations, in 70 patients. Patients with missense mutations were then divided into 2 groups: the "SHBG-region" mutation group (27 patients) and the "non-SHBG" group (24 patients). The multivariable binary logistic regression analysis showed that mutation position in the SHBG region of protein S is an independent risk factor for thrombosis in deficient patients (OR, 5.17; 95% CI, 1.29-20.65; P = .02). The patients with a mutation in the SHBG-like region also developed a thrombotic event at a younger age compared to the "non-SHBG" group in the Kaplan-Meier analysis (median thrombosis-free survival of 33 vs 47 years, respectively; P = .018). Conclusion: Our findings show that a missense mutation located in the SHBG-like region may contribute to higher thrombotic risk rather than a missense mutation located elsewhere in the protein. However, as our cohort was relatively small, these findings should be taken with this limitation.

Fibrinogen Sumperk II: dysfibrinogenemia in an individual with two coding mutations.

Fibrinogen­a 340-kDa glycoprotein­plays a crucial role in blood coagulation, platelet aggregation, wound healing, and other physiological processes. A mutation in fibrinogen may lead to congenital dysfibrinogenemia,a rare disease characterized by the functional deficiency of fibrinogen. About 580 cases of abnormal fibrinogens have been reported worldwide; thereof 335 cases in the fibrinogen Aa chain[1]. To our knowledge, only five cases of abnormal fibrinogens with two mutations [2­6] and one case of two different mutations in the same family [7] have been described earlier. A 52-year-old female was examined for bleeding. Routine hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Functional testing revealed prolonged fibrin polymerization, prolonged lysis of the clot, abnormal fibrin morphology,and fibrinopeptides release. Genetic analysis showed two heterozygous nonsense mutations­previously described mutation AaGly13Glu and a novel mutation Aa Ser314Cys. The mutation Aa Gly13-Glu was found in her brother and niece, but there was no evidence in either of the mutation Aa Ser314Cys. While mutation Aa Gly13Glu is responsible for abnormal fibrinopeptide release and prolonged thrombin time, the novel mutation Aa Ser314Cys seems to affect fibrin morphology and fibrinolysis.

Manifestation of atypical hemolytic uremic syndrome caused by novel mutations in MCP.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in genes encoding regulators of the alternative complement pathway (CFH, MCP, C3, CFI, CFB, THBD, and CFHR1-5) are connected with this disease. Polymorphisms (SNPs) in these genes might also influence the manifestation of aHUS. We have analyzed the genes of CFH, CFI, MCP, and C3 in a cohort of 10 unrelated Czech patients with clinically diagnosed familial aHUS. Surprisingly, 4 patients had mutations only in MCP, without mutations in any of the other genes that cause aHUS. Mutations, as yet unpublished, were widely distributed over the gene (SCR2 domain, signal peptide, and cytoplasmic region). The phenotype of the patients and their close relatives (14 individuals) was also investigated. Functional examination of MCP was also provided and proved lower expression on granulocytes in all mutations. Severity of disease varied, but onset was never earlier than 5 years of age. Penetrance of disease was 50% among carriers. We found that the severity and recurrence of the disease within families varied and might also be dependent on SNPs. Mutations in the MCP gene seems to be a common etiology of aHUS in Czech patients.

Hereditary thrombotic thrombocytopenic purpura and COVID-19: Impacts of vaccination and infection in this rare disease.

Introduction: Severe COVID-19 is associated with an important increase of von Willebrand factor and mild lowering of ADAMTS13 activity that may, in the presence of a strong inflammatory reaction, increase the risk of acute thrombotic thrombocytopenic purpura (TTP). Although acute episodes of immune-mediated TTP associated with COVID-19 or SARS-CoV-2 vaccination have been reported, data about clinical evolution of hereditary TTP (hTTP) during the pandemic are scarce. Method: We conducted a survey among adult patients of the International Hereditary TTP Registry about SARS-CoV-2 vaccination, COVID-19, and occurrence of acute hTTP episodes. Results: Of 122 adult hTTP patients invited to participate, 86 (70.5%) responded. Sixty-five had been vaccinated (75.6%), of which 14 had received in addition a booster, resulting in 139 individual vaccine shots. Although vaccinations in patients on plasma prophylaxis were done within 1 week of the last plasma infusion, all 23 patients treated with plasma on demand were vaccinated without prior plasma infusions. One patient on uninterrupted weekly plasma infusions presented within 3 days from his second vaccination with neurological symptoms and computed tomography scan 9 days later showed subacute ischemic/hemorrhagic frontal lobe infarction. A second male patient developed acute myocarditis after his second dose of mRNA-1273 vaccine. Twelve (14%) patients had COVID-19, associated with an acute hTTP episode in three of them: one patient had a transient ischemic attack, one a stroke, and a pregnant woman was hospitalized to intensify plasma treatment. Discussion: The risk of an acute episode triggered by COVID-19 seems higher than following vaccination in hTTP patients, who can be safely vaccinated against SARS-CoV-2.

Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A.

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.

Two novel fibrinogen variants in the C-terminus of the Bß-chain: fibrinogen Rokycany and fibrinogen Znojmo.

Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Congenital hypofibrinogenemia is a rare autosomal bleeding disorder, either recessive or dominant, characterized by a low fibrinogen plasma level. A 28-year-old asymptomatic woman (fibrinogen Rokycany) and a 54-year-old man with thrombosis and pulmonary embolism (fibrinogen Znojmo) were investigated for a suspected fibrinogen mutation after abnormal coagulation tests results were obtained. DNA sequencing showed the heterozygous point mutation Bß Asn351Lys in fibrinogen Rokycany and the heterozygous point mutation Bß Arg237Ser in fibrinogen Znojmo, respectively. The kinetics of fibrinopeptide release was found to be normal in both cases. Fibrinolysis was impaired in the Znojmo variant. The average fibril diameters of Znojmo fibrin was slightly increased, but not differing significantly from normal; formed by less fibrils with abrupt fibril terminations. Rheological studies revealed a softer clot. Rokycany fibrin was formed by significantly narrower fibrils than normal fibrin; and the clot was denser than the control clot. Rheological studies revealed a stiffer clot. Impaired fibrinolysis and abnormal clot morphology may be the cause of thrombotic episodes in the patient with Znojmo mutation. New cases of hypofibrinogenemia and dysfibrinogenemia, found by routine coagulation testing, were genetically identified as a novel fibrinogen variants Bß Asn351Lys (fibrinogen Rokycany) and Bß Arg237Ser (fibrinogen Znojmo), respectively.

Patients with acute coronary syndromes have low tissue factor activity and microparticle count, but normal concentration of tissue factor antigen in platelet free plasma: a pilot study.

OBJECTIVES: Tissue factor (TF) is a main initiator of coagulation cascade. Its determination in conditions of acute coronary syndrome is logistically difficult. Hence, in our study, the activity and the concentration of TF and the count of microparticles in the platelet free plasma (PFP) were determined. METHODS: Blood was drawn from both coronary sinus and femoral vein circulation in a cohort of 40 patients. TF activity was measured by activation of factor X in the presence of factor VIIa, whereas microparticles were detected using flow cytometry. TF antigen concentrations were determined using the ELISA test. RESULTS: TF activity in the stable angina subgroup was not significantly different from the control group (18.12 +/- 3.35 mOD/min vs. 17.72 +/- 4.05 mOD/min, respectively), but it was significantly lower in the unstable angina (7.62 +/- 4.19 mOD/min) and myocardial infarction (MI) (3.56 +/- 3.85 mOD/min) subgroups (P < 0.05). Results from the coronary sinus and femoral vein circulations were not significantly different. The count of microparticles decreased according to the severity of the acute coronary syndrome: control group, 520 +/- 172; stable angina subgroup, 532 +/- 167; unstable angina subgroup, 392 +/- 142; and MI subgroup, 165 +/- 30 (P < 0.05). There were no significant differences in concentrations of TF antigen in four subgroups. CONCLUSIONS: These results suggest that the procoagulant TF-bearing microparticles could be recruited from PFP by interaction with platelets and blood cells in the conditions of acute coronary syndrome.