Bleeding symptoms in 27 Iranian patients with the combined deficiency of factor V and factor VIII

Inherited deficiency of factors V and VIII is the most frequent combined coagulation defect. The cases reported so far are mostly single cases or small series from different centres, making it difficult to evaluate the overall pattern of clinical manifestations of the combined defect. We examined at a single institution 27 Iranian patients. Mucocutaneous and post-surgical bleeding were the most frequent clinical manifestations. The presence of two defects did not make the severity of bleeding greater than that expected in patients with single coagulation defects of similar degrees.     

Potency and mass of factor VIII in FVIII products

Summary.  Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity-based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin-free full-length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full-length rFVIII) at 1 U mL⁻¹ showed potency similar to that of the 0.7 n m (1 U mL⁻¹) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A–C and E varied over a wide range (3900–13 200 U mg⁻¹) and was higher for most lots when compared with the standard (5000 U mg⁻¹), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200–4800 U mg⁻¹). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.     

Activation of 125I-Factor IX and 125I-Factor X: Effect of Tissue Factor and Factor VII,Factor Xa and Thrombin

Activation of Factor IX and Factor X was studied by adding ¹²⁵I-Factor IX or ¹²⁵I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor X_a produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added ¹²⁵I-Factor IX or ¹²⁵I-Factor X, tissue factor and Ca2+ions, the percentage of total radioactivity in the heavy chain peak of ¹²⁵I-IX_a and the heavy chain peak of ¹²⁵I-X_a increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of ¹²⁵I-Factor IX and percent cleavage of ¹²⁵I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Facor IX by the tissue factor-Factor VII reaction product represents a physiologically significant step in normal haemostasis.     

Longitudinal study on activated factors XII and VII levels during normal pregnancy

Levels of activated factor XII (FXIIa) and VII (FVIIa) were determined in 100 women with uneventful pregnancies. Samples were divided into five study intervals: three during pregnancy, one at delivery and one 3 d postpartum.
The median (range) for FXIIa levels were 3.4 ng/ml (1.2–9.1) from 11 to 20 weeks, 4.6 ng/ml (1.4–15.2) from 21 to 30 weeks, 5.4 ng/ml (1.9–14.3) from 31st week to delivery, 5.2 (1.3–11.4) at delivery and 4.3 (1.8–8.5) ng/ml in the postpartum sample. For FVIIa the median and range levels for the five periods were 4.9 (1.7–77.3), 7.2 (2.5–80.4), 11.1 (2.9–90.6), 12.0 (3.1–64.1) and 8.2 (4.0–23.5) ng/ml. Although the increase of FVIIa was higher than that of FXIIa during pregnancy, the overall changes of FXIIa and FVIIa were highly correlated ( P  < 0.0001). At each time period the changes of FVIIa correlated with FVII:C which was not the case with FVII:Ag. These data indicate that during pregnancy both the contact phase and extrinsic pathway are activated.     

Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function

The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.     

Coagulopathy in amyloidosis: Combined deficiency of factors IX and X

Combined severe deficiencies of blood clotting factors IX and X were observed in 2 patients who suffered from systemic amyloidosis. This unique deficiency state was marked by refractoriness to Vitamin K as well as to transfusion therapy. Increased antithrombin activity was present in both individuals and corresponded in time to the emergence of a monoclonal IgG kappa light chain paraprotein in 1. Both patients demonstrated profound bleeding disorders. It is hypothesized that the Vitamin K dependent factors have special affinity for amyloid deposits due to an unusual amino acid (γ-carboxyglutamic acid) present in these factors.     

Assessing patients' and caregivers' perspectives on stability of factor VIII products for haemophilia A: a web‐based study in the United States and Canada

Haemophilia A is a rare inherited bleeding disorder characterized by an inability of the blood to clot normally. Patients can experience spontaneous or trauma‐induced joint and soft tissue bleeding and must keep coagulation factor VIII (FVIII) accessible at all times; thus, FVIII product storage and stability are critical. Our primary objective was to assess haemophilia A patients' and caregivers' experiences and preferences with FVIII product storage and stability. A secondary objective was to evaluate the use of the social media site Facebook in recruitment. In this cross‐sectional study, 145 English‐speaking adult patients and caregivers of children with haemophilia A were recruited through two state‐based haemophilia organizations in the United States (US) and one national organization in Canada for a web‐based survey assessing demographics and FVIII product ordering, usage, and storage practices. Of the 101 individuals who completed the survey, 60% resided in Canada; 57% were recruited through Facebook. Caregivers and patients responded similarly to questions about ordering practices and product usage, with some distinction between groups in storage practices. Two‐thirds of participants noted challenges with storing FVIII products, especially storage away from home. More than half preferred storing FVIII products at room temperature vs. in the refrigerator for long periods of time. FVIII product accessibility, usage and storage affect disease management. Results support the need for more convenient and accessible FVIII products for patients in daily life and while travelling. In addition, the use of social media has potential value in recruiting this population.     

Trefoil factor与消化性溃疡

Trefoil factor(TFFs)是一族由一个或者几个三叶状结构域(trefoil domain)组成的黏蛋白相关肽。自从首个三叶肽(胰解痉肽,pSP,pTFF2)于本世纪70年代末被分离以后,对其研究逐渐深入,其研究焦点集中在对胃肠道黏膜损伤后保护作用和促进溃疡愈合作用方面。同时溃疡愈合是一复杂过程,有众多因素参与,本文就有关内容做一综述。     

Intravascular recovery of VWF and FVIII following intraperitoneal injection and differences from intravenous and subcutaneous injection in mice

Intravenous infusion studies in humans suggest that both von Willebrand factor (VWF) and factor VIII (FVIII) remain intravascular in contrast to other coagulation proteins. We explored whether infusion of VWF and FVIII by either intraperitoneal (i.p.) or subcutaneous (s.c.) injection would result in efficient absorption of these large proteins into the vascular circulation. FVIII(null) or VWF(null) mice were infused with plasma-derived or recombinant VWF and/or FVIII by i.p., s.c., or intravenous (i.v.) injection. Both VWF and FVIII were absorbed into the blood circulation after i.p. injection with a peak between 2 and 4 h at levels similar to those observed in mice infused intravenously. In contrast, neither VWF nor FVIII was detected in the plasma following s.c. injection. Although i.v. injection achieved peak plasma levels quickly, both human VWF and FVIII rapidly decreased during the first 2 h following i.v. injection. Following both i.v. and i.p. infusion of VWF, the multimeric structure of circulating VWF was similar to that observed in the infusate. These results demonstrate that both VWF and FVIII can be efficiently absorbed into the blood circulation following i.p., but not s.c. injection, indicating that i.p. administration could be an alternative route for VWF or FVIII infusion.     

Probable regulation of factor VIIa–tissue factor and prothrombinase by factor Xa–TFPI and TFPI in vivo

Given that factor VIIa–tissue factor (TF) probably initiates coagulation in vivo , this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1+2 in plasma (the latter as an index of prothrombinase activity in vivo ). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa–TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa–TFPI inactivates factor VIIa–TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1+2, whereas we developed an ELISA to quantify factor Xa–TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa–TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1+2 increased in parallel in the plasmas of up to 145 healthy adults assayed ( P  = 0.007), as did the concentrations of factor VIIa and TFPI ( P  = 0.0039), and prothrombin fragment 1+2 and TFPI ( P  = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa–TFPI and factor VIIa ( P  <0.0001) and free factor Xa–TFPI and prothrombin fragment 1+2 ( P =0.0095). These results are consistent with factor Xa–TFPI regulating factor VIIa–tissue factor and prothrombinase in vivo .     

Response to desmopressin of factors XI, X and V in patients with factor VIII deficiency and von Willebrand disease

Desmopressin [1-deamino-8-d-arginine vasopressin (DDAVP)] has been successfully used in the treatment of type 1 von Willebrand disease (VWD) and mild haemophilia A (MHA). Data suggest that DDAVP can increase factor XI (FXI) plasma levels and may represent an effective treatment for mild FXI deficiency. We assessed the DDAVP response of FXI coagulant activity (FXI:C), FXI antigen (FXI:Ag), factor V coagulant activity (FV:C), and factor X coagulant activity (FX:C) in 33 individuals with VWD or MHA. DDAVP did not produce a clinically significant increase in FXI:C, FXI:Ag, FX:C or FV:C in any patient. The mean +/- SD FXI:C pre-DDAVP (time 0) and at 1 h post-DDAVP was 90.7 (+/-22.9) U/dl and 92.1 (+/-20.9) U/dl, respectively. The mean (+/-SD) FXI:Ag at time 0 and 1 h was 92.2 (+/-20.1) U/dl and 89.9 (+/-21.3) U/dl, respectively. There was a small reduction at 1 h post-DDAVP in both FV:C, from 101.8 (+/-20.9) U/dl to 97.2 (+/-21.4) U/dl (P < 0.001), and FX:C from 103 (+/-19.5) U/dl to 98.8 (+/-18.7) U/dl (P < 0.001). No significant increase in FXI:C, FXI:Ag, FV:C or FX:C levels was seen at 4 h post-DDAVP. This study failed to demonstrate a clinically significant increase in the levels of FXI, FX or FV following administration of DDAVP.     

Evidence for relationships between antiperinuclear and IgG rheumatoid factor

Summary Antiperinuclear factor (APF) and IgG-rheumatoid factor (IgG-RF) has been found in 64% and 48% of cases of rheumatoid arthritis, 36% and 50% of cases of psoriasis and 31% and 45% of cases of primary Sjögren's syndrome. A close relationship between APF and IgG-RF is suggested by statistical and experimental data. Purified IgG-RF has some degree of APF activity.     

Differential clot stabilising effects of rFVIIa and rFXIII-A2 in whole blood from thrombocytopenic patients and healthy volunteers

The haemostatic effect of recombinant activated factor VII (rFVIIa;NovoSeven) in thrombocytopenic patients has been a matter of controversy. Haemostasis by rFVIIa occurs via FVIIa-mediated thrombin generation in a platelet-dependent manner and may therefore be suboptimal in patients without functional platelets. Under such conditions, a clot-stabilizing agent, such as factor XIII (FXIII), may supplement the effect ofrFVIIa and improve haemostasis. Recombinant factor XIII (rFXIII-A2) is produced as an A2 homodimer of the FXIII A subunit and is equivalent to cellular FXIII normally found in platelets. The combined effects of rFVIIa andrFXIII-A2 were evaluated in clot lysis assays using factor XIII-deficient plasma and by whole blood thrombelastography (TEG) analysis from normal donors and thrombocytopenic stem cell transplantation patients. Clotting time was shortened by rFVIIa (0.6-10 microg/ml). rFVIIa only modestly improved anti-fibrinolysis,whereas rFXIII-A2 (0-20 microg/ml) enhanced anti-fibrinolysis without effect on clotting time. TEG analysis showed rFVIIa shortened the clotting time, and enhanced clot development, maximal mechanical strength and resistance to fibrinolysis, whereas, rFXIII-A2 enhanced clot development,maximal mechanical strength and markedly enhanced resistance to fibrinolysis. These data illustrate that rFVIIa and rFXIII-A2 contribute to clot formation and stability by different mechanisms suggesting enhanced haemostatic efficacy by combining these agents.     

Pharmacokinetics of factor VIII and factor IX

Summary.  A survey of principal pharmacokinetic (PK) studies on factor VIII (FVIII) and factor IX (FIX) plasma- and rDNA-derived concentrates, analysed by means of the PKRD program, has been performed. Notwithstanding the accurate definition of the study design, released in 1991 by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC-ISTH), a large variability of PK parameters has been pointed out. In the majority of the PK studies, the size of the population is small. In this situation, a careful individualization of haemophilia therapy is strongly recommended. The tailored prediction of loading and maintenance dosages and the need for strict control of trough FVIII/IX levels are mandatory not only to decrease the risk of bleeds but also to spare financial resources. Recently, the old problem of FVIII assay standardization has again become a concern among physicians, especially after the introduction of B-domain deleted rFVIII concentrate. The discrepancies between the widely used one-stage clotting assay and the chromogenic substrate assay seem to be solved by the introduction of a product-specific laboratory standard.     

Combined deficiency of factor V and factor VIII. A report of another case

Summary A patient with combined factor V and factor VIII deficiency is presented. The bleeding manifestations were: easy bruising, post-traumatic bleeding, bleeding after tooth extractions. The main laboratory feature was a prolonged partial thromboplastin time which was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum, hemophilia A plasma or plasma of another patient with combined factor V and factor VIII deficiency. The thromboplastin generation test was clearly abnormal and was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum. Prothrombin consumption was also defective.Prothrombin time was slightly prolonged too, Thrombin time, platelet and vascular tests were within normal limits and there was no hyperfibrinolysis. Factor VIII was 8% of normal, whereas factor V was 14% of normal. Factor VIII associated antigen was normal. All other clotting factors were within normal limits.The parents of the propositus were consanguineous (first cousins) but had normal factor V and factor VIII activity and normal factor VIII antigen. The same was true for other family members. The hereditary transmission of the condition appears autosomal recessive.This study was supported in part by a grant from the C.N.R. (grant CT. 74.00189.04).     

Growth Factors and Diabetic Neuropathy

A variety of soluble growth factors influence the peripheral nervous system. Although of considerable importance during development and growth, they appear also to be implicated in tissue maintenance in adult life and, particularly, during nerve regeneration. In addition, cell-surface and extracellular connective tissue matrix molecules are intimately involved in regeneration. So far, the possible participation of such growth factors in the causation of diabetic neuropathy is only speculative, but there are indications that their use could be of value in treatment.     

Effect of glycoPEGylation on factor VIIa binding and internalization

Summary. Recombinant coagulation factor VIIa (rFVIIa), which is widely used for treatment of bleeding episodes in haemophilia patients with inhibitors, is cleared from the circulation relatively fast with a plasma half‐life of 2–4 h. PEGylation is an established and clinically proven strategy for prolonging the circulatory life‐time of bio‐therapeutic proteins. The aim of this study was to investigate the effect of glycoPEGylation of rFVIIa on rFVIIa binding to its cellular receptors and its subsequent internalization. rFVIIa and glycoPEGylated rFVIIa were labeled with ¹²⁵I and the radio‐iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells and fibroblasts. FVIIa‐TF activity at the cell surface was analyzed by a factor X activation assay. Modification of rFVIIa with PEG impaired rFVIIa binding to both endothelial cell protein C receptor and tissue factor (TF) on cell surfaces. The internalization of PEGylated rFVIIa in endothelial cells and fibroblasts was markedly lower compared to the internalization of rFVIIa in these cells. PEGylated rFVIIa was able to activate factor X on TF expressing cell surfaces at a rate similar to that of unmodified rFVIIa when the cells were not subjected to multiple washings to remove the free ligand. General effects such as steric hindrance or changes in electrostatic binding properties of the modified rFVIIa to its receptors are probably responsible for this impairment rather than a loss of specific recognition of the receptors, which could explain near normal activation of factor X by glycoPEGylated rFVIIa on TF expressing cells while its uptake is reduced.