Fifth Åland Island conference on von Willebrand disease.

The fifth Åland Island meeting on von Willebrand disease (VWD) was held on the Åland Islands, Finland, from 22 to 24 September 2016-90 years after the first case of VWD was diagnosed in a patient from the Åland Islands in 1926. This meeting brought together experts in the field of VWD to share knowledge and expertise on current trends and challenges in VWD. Topics included the storage and release of von Willebrand factor (VWF), epidemiology and diagnostics in VWD, treatment of VWD, angiogenesis and VWF inhibitors.

An increased tendency in fibrinogen activity and its association with a hypo-fibrinolytic state in early stages after injury in patients without acute traumatic coagulopathy (ATC).

Acute traumatic coagulopathy (ATC) diagnosed by prolongation of APTT and/or PT/INR involves alterations in platelet activity, coagulation and fibrinolysis. However, data showing the haemostatic situation in injured patients without ATC are scarce. To assess whether haemostatic impairment is also present in injured patients without ATC, ten injured patients without ATC and ten normal individuals were examined. The patients were sampled on arrival at the emergency department 0, 2, 12 h after surgical or other intervention. Thrombin generation, fibrin formation and fibrin proteolysis were determined via several laboratory methods, using tissue factor as the coagulation trigger. Thrombograms demonstrated that trauma accelerated both thrombin generation and decay. In the presence of unaffected peak thrombin levels, these two contradictory effects cancelled each other out, leading to the global endogenous thrombin potential (ETP) remaining normal. Under the mediation of normal ETP, fibrin network permeability (Ks) kept the reference levels in the two groups of subjects. Fibrinogen (FBG) activity (Clauss) rose with time from 0 to 2 h and 12 h, which significantly slowed down Clot Lysis Potential as determined by an in vitro method with exogenous t-PA. SUMMARY: the main haemostatic impairment in the present patients concerned an increased tendency in FBG activity. Since an increase in FBG is a biomarker of acute inflammation and also predicts greater fibrin production which down-regulates fibrinolysis, we suggest that during early stages after injury, patients without ATC may suffer from worsening inflammation and confront enhancement of thrombosis risk due to dysfunction of fibrinolysis.

Fibrinogen depletion after plasma-dilution: impairment of proteolytic resistance and reversal via clotting factor concentrates.

In trauma patients, resuscitation treatment of intravascular volume may cause haemodilution including blood cell- and plasma-dilution. After plasma-dilution, fibrinogen is the first factor that decreases to critically low concentrations. Fibrin formed in lowered levels is susceptible to fibrinolysis, a natural forerunner for bleeding. To assess whether a fibrinogen concentrate or a factor XIII (FXIII) concentrate can reverse the impairment of fibrin properties after plasma dilution, different laboratory methods were used to determine thrombin generation and fibrin quantity/quality in a normal plasma sample diluted in vitro. Coagulation and clot lysis by plasmin were triggered with tissue factor and rt-PA, respectively.We found that while the endogenous thrombin potential (ETP) was unaffected after plasma-dilution due to postponement of thrombin decay, levels of fibrinogen and hence fibrin were decreased in dilution degree-dependency. The imbalance between influence of the dilution on thrombin activity and fibrin formation brought unexpected outcomes of fibrin properties: the formed clots favoured the degradation by plasminbut the fibrin networks remained tighter/less permeable. This proteolytic tendency was partly overturned by the fibrinogen concentrate added (total fibrinogen ≥ 2 g/l), and much more affected if used in combination with tranexamic acid (a fibrinolysis inhibitor) at small doses. No reversal effect resulted from the FXIII concentrate added. We conclude that plasma-dilution did reduce the proteolytic resistance of formed clots. The fibrinogen concentrate, better together with small doses of tranexamic acid, may reverse the impairment of fibrin property.The FXIII concentrate is not effective in this regard in our in vitro model using platelet-poor plasma.

Decreased fibrin network permeability and impaired fibrinolysis in the acute and convalescent phase of ischemic stroke.

INTRODUCTION: We investigated fibrin network permeability and fibrinolysis in the acute and convalescent phase of ischemic stroke. METHODS: 20 patients with a mean age of 74 years were studied in the acute (day 1) and convalescent phase (day 60) of ischemic stroke. 23 healthy individuals (controls) were also investigated. Fibrin formation in the samples was triggered by addition of tissue factor (1 pmol/L) and washed frozen-thawed platelets obtained from a healthy donor. The permeability constant (K(s)), which reflects fibrin network permeability, was then calculated from liquid flow measurements. A global assay newly developed in our group was also employed to determine the balance between fibrin formation ("Coagulation profile"; Cp) and fibrin degradation ("Fibrinolysis profile"; Fp) in the same samples. We also measured PAI-1 antigen and fibrinogen concentrations in plasma. RESULTS: As compared to controls, the stroke patients had lower Ks (lower fibrin network permeability) both on day 1 and on day 60 (p < 0.01 and p < 0.05, respectively). Fibrinolysis, assessed by Fp, was reduced on both day 1 and day 60 (p < 0.001, compared to controls), and PAI-1 concentrations were increased (p < 0.01 for both, compared to controls). Fibrin formation capacity in plasma (i.e. Cp) was increased in the acute phase (p < 0.05) but not in the convalescence, as compared to controls. CONCLUSION: The combination of a proneness to form a tighter fibrin network and impaired fibrinolysis is a feature of ischemic stroke that is present in both the acute and convalescent phase of the disease.

The plasma concentration of activated protein C appears normal in patients with haemophilia.

Increased concentration of activated protein C (APC) has been observed in patients with thromboembolic disorders, but whether the level of APC in patients with bleeding disorders is decreased remains unknown. Seventy patients with haemophilia A or B with mild, moderate or severe form were studied. Detailed information on bleeding, arthropathy and factor consumption was collected during a 10-year period. The clinical severity of the disease was expressed as the Hemophilia Severity Score (HSS). Plasma concentration of APC was measured as APC in complex with protein C inhibitor. The median concentration of APC-PCI complex in patients with haemophilia was 0.14 microg L(-1) and it did not differ between the types and forms. In 16 patients with severe haemophilia A and the inversion mutation in intron 22, there was no correlation between clinical severity and the concentration of APC-PCI complex. Patients with haemophilia appear to generate normal concentrations of APC during basal conditions. APC does not seem to be an important modulator of the phenotypic expression of haemophilia.

Validation of a composite score for clinical severity of hemophilia.

INTRODUCTION: Evaluation of modulators of the phenotypic expression of hemophilia may benefit from a scoring system that reflects several aspects of the clinical severity instead of only one dimension. METHODS: We describe here how we constructed a composite Hemophilia Severity Score (HSS) and performed validation. The items in the HSS are annual incidence of joint bleeds, World Federation of Hemophilia Orthopedic joint score, and annual factor consumption. The latter two were adjusted for age at start of prophylaxis and body weight. Data for 100 adolescent or adult patients with hemophilia A or B in the mild, moderate or severe form without inhibitors were collected for the 1990-1999 period. We evaluated the reliability (multidimension property, test-retest) and validity (content, convergent, discriminant and known groups) of the score. RESULTS: The HSS ranged from 0 to 0.94 and was higher in severe hemophilia A than severe hemophilia B (median 0.50 and 0.24; P = 0.031). The validation indicated that the HSS is reliable and reflective of the clinical severity of hemophilia. The presence of factor V G1691A or prothrombin G20210A polymorphisms was found in 13 patients. The clinical severity, measured as the HSS or each of the three components, appeared to be modified by prothrombin G20210A but not by FV G1691A. CONCLUSION: The HSS is a well-defined tool that provides a comprehensive representation of the clinical severity of hemophilia in adults. It would be useful in larger studies on the assessment of modulators of the phenotypic expression of hemophilia.

Preanalytical conditions that affect coagulation testing, including hormonal status and therapy.

Preanalytical conditions, be they due to the individual's physiologic state or to exogenous factors, can affect coagulation factors, in either a transient or a persistent manner, and need to be considered in laboratory testing. These conditions include physical and mental stress, diurnal variation, hormone levels and posture at the time of blood drawing. While testing of these factors has not been exhaustive and some results are conflicting, guidelines for testing conditions can be given.

Occurrence of cold activation of transfusion plasma during storage at +4 degrees C.

BACKGROUND AND OBJECTIVES: The storage of transfusion plasma at +4 degrees C sometimes leads to the activation of several proteolytic systems. In this study the frequency of cold activation was investigated, as well as whether cold activation of plasma is an individually recurrent property of the donor. MATERIALS AND METHODS: Plasma units prepared from whole blood obtained from 100 male donors were stored at +2 degrees to +5 degrees C, in bags for 28 days and in cryotubes for up to 42 days. Samples from plasma units, collected by apheresis from 100 male donors, were stored in cryotubes for up to 42 days. Cold activation was measured weekly as kallikrein-like activity of plasma. Samples from repeat apheresis plasma units from 32 donors were measured 12-20 months later. The effects of storage on the contact, coagulation and fibrinolytic systems were determined. RESULTS: The cumulative frequency of cold-activated plasma units stored in bags was 5% on day 7 and 18% on day 28. After 42 days in cryotubes, 49% of the plasma units were cold activated. Large intraindividual differences in the onset-day of cold activation were observed in plasma samples of some donors. During cold activation, an increase in kallikrein-like activity was accompanied by a decrease in C1 esterase inhibitor activity and an increase in the concentrations of activated factor VII and fibrinopeptide A. The functional plasminogen level was unchanged, while a minor decrease in plasmin inhibitor activity was combined with a corresponding increase in the concentration of plasmin-plasmin inhibitor complex. CONCLUSIONS: The cumulative frequency of cold-activated plasma units increased in a time-dependent manner during storage at +2 degrees to +5 degrees C for 42 days. The intraindividual onset-day of cold activation varied widely between plasma samples of some donors. Cold activation was associated with a high degree of activation of the contact and coagulation systems. The fibrinolytic system was scarcely affected.

Does an enzyme other than thrombin contribute to unexpected changes in the levels of the different forms of thrombin activatable fibrinolysis inhibitor in patients with hemophilia A, hemophilia B and von Willebrand disease?

Pro-thrombin activatable fibrinolysis inhibitor (pro-TAFI), also called plasma procarboxypeptidase B or U, is one of the modulators of fibrinolysis in blood. Pro-TAFI is activated by thrombin/thrombomodulin complex or by plasmin to a carboxypeptidase B-like enzyme (TAFI) of 35.8 kD molecular weight. TAFI spontaneously becomes inactive as a result of a temperature-dependent conformational change in the protein (TAFIi). In this study, pro-TAFI, total TAFI antigen and TAFI-TAFIi antigen levels were measured in 32 patients with hemophilia A, 4 patients with hemophilia B, 21 patients with von Willebrand disease (VWD) and 13 healthy controls. A statistically significant decrease in pro-TAFI was found in all groups (10.72+/-4.57 mg/L (p<0.001); 8.00+/-2.35 mg/L (p<0.01) and 8.98+/-2.33 mg/L (p <0.001) for hemophilia A, hemophilia B and VWD, respectively) compared to controls (17.85+4.61 mg/L). A statistically significant increase in TAFI-TAFIi antigen was found in hemophilia A (1.05+/-1.01 mg/L) (p<0.05) and in VWD patients (0.96+/-1.01 mg/L) (p<0.05) compared to controls (0.55+/-0.36 mg/L). There was no difference in total TAFI antigen levels between any group of patients and the controls. Neither did pro-TAFI nor TAFI-TAFIi levels differ within the group of hemophilia A patients in relation to severity (mild, moderate and severe) or among the VWD patients in relation to subtype (type 1, type 2A and type 3). These findings indicate an increased conversion of pro-TAFI to TAFI and/or TAFIi in patients with bleeding disorders. As thrombin generation is seriously impaired in these patients and almost absent in hemophilia A and B and in type 3 VWD, it is possible that plasmin mediates pro-TAFI activation in these patients. Enhanced fibrinolysis via generation of plasmin has previously been reported in hemophilia and VWD. Activation of pro-TAFI by plasmin may be a feedback mechanism that counterbalances increased fibrinolysis in patients with bleeding disorders. The relationship between the TAFI activation pathway and bleeding complications associated with hemophilia A, hemophilia B and VWD requires further investigation.

Increased hemostasis potential persists in women with previous thromboembolism with or without APC resistance.

BACKGROUND: Activated thrombin generation and depressed fibrinolysis due to the presence of activated protein C (APC) resistance with or without factor (F)V Leiden mutation are associated with development of deep venous thrombosis (DVT). OBJECTIVE: A better understanding of the mechanism behind the risk of recurrence of DVT, using our new, recently developed assay of overall hemostasis potential (OHP). PATIENTS AND METHODS: Levels of OHP, as well as APC resistance and FV Leiden mutation, were determined in 88 women (cases) who had previously experienced DVT in connection with pregnancy, and in 25 young healthy individuals (controls). Clotting time and clot lysis time were also investigated. RESULTS: OHP levels in the patients were increased compared with the controls. In the cases with APC resistance and the Leiden mutation this imbalance in hemostasis potential was more severe than in those without. The group with the more severe imbalance had shorter clotting times and longer clot lysis times. CONCLUSIONS: A procoagulant state perseveres in patients with a history of pregnancy-related DVT, even after the symptomatic phase is over. The mechanisms behind such an imbalance in overall hemostasis are enhanced thrombin generation and depressed fibrinolysis. These findings may underscore the need for thromboprophylaxis to prevent recurrence of thromboembolism in risk situations.

Increased plasma fibrin gel porosity in patients with Type I diabetes during continuous subcutaneous insulin infusion.

BACKGROUND: Patients with Type 1 diabetes have a tighter plasma fibrin gel structure, to which impaired glycemic control might contribute. Improved glycemic control can be achieved with continuous subcutaneous insulin infusion (CSII). OBJECTIVES: The aim of the present study was to investigate the effect of CSII on plasma fibrin gel properties and circulating markers of inflammatory activity in patients with Type 1 diabetes. PATIENTS AND METHODS: Twenty-eight patients were investigated before and after 4-6 months' treatment with CSII. Fibrin gel structure formed in vitro from plasma samples was investigated by liquid permeation of hydrated fibrin gel networks. P-fibrinogen was analyzed by a syneresis method. Comparisons were made between patients with improved (> 0.5%) and unchanged (< 0.5%) glucosylated hemoglobin (HbA1c) during CSII. RESULTS: Eighteen patients showed improved and 10 patients unchanged HbA1c during CSII. P-fibrinogen, high sensitive C-reactive protein and serum amyloid A-antigen were not significantly changed, while fibrin gel permeability (Ks) and fiber mass-length ratio ( micro ) increased in both groups (P < 0.02). P-insulin and triglycerides decreased (P < 0.05) in both groups, while reductions of total cholesterol and intercellular adhesion molecule-1 were seen only in patients with improved HbA(1c) (P < 0.05). Absolute changes in Ks were inversely correlated to changes in plasma fibrinogen (r = 0.50; P < 0.01) and in LDL-cholesterol (r = 0.46; P < 0.05). CONCLUSIONS: Treatment with CSII in patients with Type 1 diabetes is associated with increased plasma fibrin gel porosity. Slight attenuation of the inflammatory activity was also observed. The changes in fibrin gel porosity seem to be mainly mediated by changes in plasma fibrinogen and blood lipids, and are probably secondary to improved insulin sensitivity.

The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX.

Patients with hemophilia have an impaired thrombin generation and therefore form loose fibrin hemostatic plugs that are easily dissolved by fibrinolysis. This prevents maintained hemostasis in these patients, resulting in a severe bleeding disorder. Recombinant (F)VIIa has been shown to enhance thrombin generation on already thrombin-activated platelets in the absence of FVIII and FIX. An efficacy rate of 80-90% has been found in hemophilia patients with inhibitors against FVIII or FIX both in association with major surgery and in the treatment of serious bleedings. In a model measuring fibrin clot permeability in a platelet-containing system described by Blombäck et al. (1994) this was demonstrated to be dependent on the concentration of FVIII and FIX. The addition of rFVIIa in concentrations of 1.9, 4.8 and 9.6 microg mL(-1) normalized fibrin clot permeability. The concentration of 1.9 microg mL(-1) of rFVIIa normalized clot permeability in this system and the higher concentrations of rFVIIa added only slightly to the effect. No further decrease in clot permeability was found when rFVIIa in a concentration of 1.9 microg mL(-1) was added to a sample with a normal concentration (100%) of FVIII or FIX. Higher concentrations of rFVIIa added to the plasma containing 100% of FVIII or FIX induced only a slight further decrease of fibrin permeability constant, arguing against any unwanted effect of extra rFVIIa on clot permeability in the case of a normal hemostasis. Furthermore, the fibrin network was studied with 3D microscopy and the loose network found in the absence of FVIII or FIX increased in density with increasing FVIII or FIX concentrations. The addition of rFVIIa to FVIII- or FIX-deficient systems altered the network structure, making the fibers thinner and more tightly packed.

Nicotinamide is a potent inhibitor of proinflammatory cytokines.

The present study investigates the modulating effects of nicotinamide on the cytokine response to endotoxin. In an in vitro model of endotoxaemia, human whole blood was stimulated for two hours with endotoxin at 1 ng/ml, achieving high levels of the proinflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF alpha. When coincubating whole blood, endotoxin and the vitamin B3 derivative nicotinamide, all four cytokines measured were inhibited in a dose dependent manner. Inhibition was observed already at a nicotinamide concentration of 2 mmol/l. At a concentration of 40 mmol/l, the IL-1 beta, IL-6 and TNF alpha responses were reduced by more than 95% and the IL-8 levels reduced by 85%. Endotoxin stimulation activates poly(ADP-ribose)polymerase (PARP), a nuclear DNA repair enzyme. It has been hypothesized that the anti-inflammatory properties of nicotinamide are due to PARP inhibition. In the present study, the endotoxin induced PARP activation was dose dependently decreased with 4-40 mmol/l nicotinamide or 4-100 micro mol/l 6(5H) phenanthridinone, a specific PARP inhibitor. 6(5H)phenanthridinone however, failed to inhibit the proinflammatory cytokines. Thus, the mechanism behind the cytokine inhibition in our model seems not to be due to PARP inhibition. In conclusion, the present study could not only confirm previous reports of a down-regulatory effect on TNFalpha, but demonstrates that nicotinamide is a potent modulator of several proinflammatory cytokines. These findings demonstrate that nicotinamide has a potent immunomodulatory effect in vitro, and may have great potential for treatment of human inflammatory disease.

Overall haemostatic potential can be used for estimation of thrombin-activatable fibrinolysis inhibitor-dependent fibrinolysis in vivo and for possible follow-up of recombinant factor VIIa treatment in patients with inhibitors to factor VIII.

Thrombin generation induced by recombinant factor VIIa (rFVIIa) in patients with haemophilia and/or inhibitors to factor VIII/IX could enhance generation of thrombin-activatable fibrinolysis inhibitor (TAFI), a recently described link between coagulation and fibrinolysis. TAFI is unstable and it is not easy to measure its active form in vivo. Overall haemostatic potential (OHP) is a novel method for haemostasis estimation, based on determination of the fibrin aggregation curve in which tiny amounts of thrombin are used for activation of clotting. We measured OHP in six patients with inhibitors to factor VIII before injection of rFVIIa and 10 and 120 min thereafter. Overall fibrinolytic potential (OFP) and clot lysis time (CLT) analysed by this method could be used for indirect estimation of TAFI generation. We found no change in pro-TAFI and total TAFI antigen before and after treatment with rFVIIa. OHP was almost undetectable before treatment but increased into the range of normal pooled plasma 10 and 120 min after rFVIIa treatment, as did CLT. However, after addition of potato tuber carboxypeptidase inhibitor, a specific inhibitor of TAFI, the shortening of CLT was lower than that in NPP. OFP was increased in patient plasma both 10 and 120 min after treatment compared with NPP. There was a strong positive correlation between pro-TAFI concentration and shortening of CLT after PTCI addition and a negative correlation between pro-TAFI concentration and OFP 10 min after rFVIIa injection. Thus, rFVIIa normalizes OHP and CLT 10 min after injection. While this improvement slightly decreases, but still exists after 2 hours, it suggests efficacy in bleeding prevention using a protocol based on rFVIIa administration every 2 hours.

Does thrombin activatable fibrinolysis inhibitor (TAFI) contribute to impairment of fibrinolysis in patients with preeclampsia and/or intrauterine fetal growth retardation?

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a relatively recently described glycoprotein (MM 55 KDa) that can be converted into its active form by the thrombin/thrombomodulin complex and potentially inhibits fibrinolysis. Since it represents a link between coagulation and fibrinolysis, TAFI can be expected to play a part in various clinical conditions associated with a thrombotic tendency. Preeclampsia (PE) and intrauterine fetal growth retardation (IUFGR) are fairly common complications of pregnancy that are characterized by hemostatic abnormalities. TAFI antigen and its influence on hemostasis was investigated in 46 women with PE and/or IUFGR and in 16 normal pregnancies. We found a significant decrease of TAFI antigen in the patient group. Using the recently described method Overall Hemostatic Potential (OHP) in plasma we measured clot lysis time (CLT) and overall fibrinolytic potential (OFP). We found that CLT is prolonged and OFP decreased in patients with PE and/or IUFGR. Since OFP did not increase after addition of the specific inhibitor of TAFI (potato tuber carboxypeptidase inhibitor), it seems that TAFI does not contribute to the impairment of fibrinolysis in these patients. Since serum albumin was decreased together with presence of proteinuria and aminotransferases were increased in the patients, it seems that one explanation for the decrease in TAFI could be reduced hepatic synthesis and an increased loss in urine. It an be speculated that this mechanism can prevent more serious thrombotic complications in patients with PE and/or IUFGR.