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.

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.

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.

HTLV-III infection in homosexuals and hemophiliacs in Sweden.

Two hundred and three homosexual (HS) men and 114 hemophiliacs in Sweden were examined for serum antibodies to human T-lymphotropic virus type III (HTLV-III) and for alterations of T-lymphocyte subsets. Sera were screened for HTLV-III antibodies by an enzyme-linked immunosorbent assay and/or a dot immunobinding assay, and positive reactions were confirmed by Western blotting. HTLV-III antibodies were demonstrated in 13 of 13 (100%) HS men with acquired immune deficiency syndrome, in 63 of 67 (94%) HS men with persistent generalized lymphadenopathy, in 17 of 45 (38%) symptomatic HS men, and in 6 of 78 (8%) asymptomatic HS men but in none of 108 male blood donors. Seropositive HS men had significantly lower T4/T8 (helper/suppressor) cell ratios and T4 cell numbers than had seronegative HS men. Seronegative HS men had decreased T-cell ratios compared to controls but not decreased T4 cell numbers. Among hemophilia A patients, HTLV III antibodies were demonstrated in 40 of 48 (83%) cases treated with American factor VIII concentrate and in 17 of 29 (59%) cases treated with both American and Swedish concentrates but in none of 13 cases treated exclusively with Swedish factor VIII. Twenty-one hemophilia B patients treated with Swedish factor IX concentrates were all seronegative, whereas one of 3 hemophilia B cases treated with imported factor IX was seropositive. T4/T8 cell ratios were significantly lower in seropositive as compared to seronegative hemophilia A patients.

Combination of activated protein C resistance and antibodies to phospholipids in the development of thrombosis.

An insufficient response to activated protein C (APC) resistance and antibodies against phospholipids (PLa) are frequent laboratory findings associated with thrombosis. Studies investigating the coexistance of these two factors in thrombophilic patients and in patients with autoimmune disorders are summarized. The investigation of thrombophilic patients has revealed PLa in 35%. About half of these patients had a combination of PLa and APC resistance proved by the Arg506-Gln mutation in factor V (FV). A combination of APC resistance with PLa in this group of patients did not increase the risk of thrombosis recurrence. In the PLa-positive patients with autoimmune disorders having thrombosis in 10% to 56%, the incidence of the Arg506-Gln mutation in FV was significantly lower and varied between 2% and 9%. A known ability of PLa to induce APC resistance experimentally was often a reason to avoid the determination of APC response in PLa-positive patients. We suggest that PLa may play a dual role in the development of thrombosis in the carriers of the Arg506-Gln mutation in FV. On the one hand, PLas favor thrombosis by maintaining the APC response at a constantly low level. On the other hand, preventing the interaction of coagulation proteins on the phospholipid surface PLa may protect them from hypercoagulation.

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.

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.

Injection of xenogeneic endocrine pancreatic tissue into the portal vein--effects on coagulation, liver function, and hepatic hemodynamics. A study in the pig-to-dog model.

The safety of injecting discordant xenogeneic fetal endocrine pancreatic tissue into the portal vein was studied in a pig-to-dog system. It was found that minced fetal porcine pancreas and fetal porcine isletlike cell clusters prepared by collagenase digestion and culture could be injected with only minor or no hepatic hemodynamic disturbances. Coagulation studies revealed a small increase in plasma fibrinopeptide A, but this increase could be prevented by heparinization of the recipient. There was no consumption of fibrinogen or platelets. In contrast, injection of minced adult porcine pancreas caused pronounced hepatic hemodynamic changes and marked coagulation abnormalities, indicating consumption coagulopathy. The present finding that fetal porcine pancreas can be injected intraportally without deleterious effects in dogs provides a foundation for the eventual clinical use of such material as treatment for insulin-dependent diabetes mellitus.

Estrogen treatment of tall girls: risk of thrombosis?

In 34 girls who were treated with large doses of ethinylestradiol because of expected excessive tall stature, antithrombin activity in the blood was followed before, during, and after treatment. All girls showed lower levels of antithrombin during treatment than before or after estrogen administration; the mean concentration during treatment was at or below the lower limit of control values (-2 SD). In three girls, the antithrombin levels were so low that the medication was withdrawn. There was no difference between the mean levels of antithrombin activity in the three groups treated with 0.25, 0.50, or 1.0 mg of ethinylestradiol. Deficiency of antithrombin is known to be associated with an increased risk of thrombosis. Therefore, antithrombin concentrations in blood should be determined before and during treatment with large doses of estrogens.

High frequency of low plasma haptoglobin values found in hemophilia A patients on prophylactic treatment with factor VIII concentrates - a sign of hemolysis?

Low plasma haptoglobin values have been observed in hemophilia A patients on regular prophylactic treatment with factor VIII concentrates. Two of 3 patients treated with fraction I-0 (Kabi) and 7 of 11 patients treated with high-purity concentrates (Hyland) had low haptoglobin values. Four of 8 patients who were treated with high-purity concentrates prescreened for a low content of anti-A and anti-B immunoglobulins still showed low haptoglobin levels. Unexpectedly, 2 patients of blood group 0 showed low haptoglobin values. The presence of irregular erythrocyte alloantibodies and/or other contaminants of the concentrates might thus also be a cause of hemolysis resulting in an increased consumption of haptoglobin. Elevated lactate dehydrogenase levels were also frequent. No correlations were found between albumin, aspartate or alanine aminotransferase levels and haptoglobin levels.

The effect of prophylaxis with low dose heparin on blood coagulation parameters. A double blind study in connection with transvesical prostatectomy.

Heparin was administered subcutaneously 5.000 IU twice daily using a double blind method to ten of twenty-one patients undergoing transvesical prostatectomy. Platelet count, APTT, throbin time, Reptilase time, Normotest, fibrinogen, Factor-VIII, ethanol gelation test, antithrombin III, fibrinolytic degradation products, alpha1-antitrypsin and alpha2-macroglobulin were studied pre- and postoperatively up to the 10th postoperative day. Statistical analysis of parameters of blood coagulation and fibrinolysis showed no significant difference between the two groups. The mechanism by which low dose heparin exerts its thromboprophylactic effect could not be elucidated from the study of the investigated parameters. The laboratory data gave no indication to a possible increased risk of postoperative hemorrhage.

Demonstration of reactivity to airborne and food allergens in cutaneous vasculitis by variations in fibrinopeptide A and other blood coagulation, fibrinolysis and complement parameters.

In a 32-year-old woman and a 40-year-old man with cutaneous vasculitis, etiological allergic responses to foods and airborne allergens were found. During provocation tests, observations were made on blood levels of fibrinopeptide A(FPA) and coagulation factors, fibrinogen degradation products (FDP) and serum complement components. Skin biopsies were taken for microscopic and immunofluorescence analysis. In case 1, anaphylactoid allergy to milk and reaginic and anaphylactoid hypersensitivity to grass pollens were found. Dermal provocations with grass pollens gave arthralgia, hematomas, serum C3 fluctuation, factor VII reduction and fibrinolysis. During peroral milk challenge, transient increases in FPA and FDP levels were observed before symptoms appeared. In case 2, anaphylactoid hypersensitivity responses to bacteria, animal danders, foods and pollens were found. Two inhalations with sheep-wool extract resulted in a typical skin eruption. The first also gave an early reduction of C3 and then FPA liberation. Nasal birch-pollen test gave an increase of FPA in the latent period and then typical nodules. At least no low molecular weight FDP were detected during provocations. In patients with vasculitis reactions to exogenous allergens, FPA and FDP estimations after provocations may discriminate harmful from innocuous allergens and reveal individual response patterns in coagulation and fibrinolysis systems.

Prediction of changes in levels of haemostatic variables during natural menstrual cycle and ovarian hyperstimulation.

To find if there is a relation between levels of haemostatic variables at low and high hormonal levels (oestradiol and progesterone) in an individual, blood samples were drawn from 12 women repeatedly during one menstrual cycle (Study I) and from 14 women undergoing in vitro fertilization, before hormonal stimulation and daily during the periovulatory period (Study II). Regression coefficients were calculated between minimum (independent) and maximum (dependent) values in both studies. In Study II highly significant regression coefficients were found between oestradiol minimum (pretreatment) and maximum (median 105 and 4730 pmol/l, respectively) for coagulation factors FVIII, von Willebrand Factor (antigen), FVII (activity and antigen), fibrinogen, protein C, protein S (free), antithrombin, plasminogen and plasminogen activator inhibitor-1; furthermore, between progesterone-minimum at day -3 or -2 (related to ovum pick up) and maximum (median 4.7 and 98 nmol/l, respectively) for FVIII, von Willebrand Factor, FVII (activity and antigen), protein C, protein S (free), and plasminogen. In Study I, where much lower hormonal levels were obtained at maximum (oestradiol median 297 pmol/l and progesterone 47 nmol/l), the same pattern was observed especially for FVII, FX, fibrinogen, plasminogen and plasmin inhibitor. Thus, the concentration of a haemostatic variable at a low oestradiol or progesterone level can predict the level at a high hormonal level.

Intensive initial oral anticoagulation and shorter heparin treatment in deep vein thrombosis.

In order to investigate whether a more intensive initial oral anticoagulation still would be safe and effective, we performed a prospective randomized study in patients with deep vein thrombosis. They received either the conventional regimen of oral anticoagulation ("low-dose") and heparin or a more intense oral anticoagulation ("high-dose") with a shorter period of heparin treatment. In the first part of the study 129 patients were randomized. The "low-dose" group reached a stable therapeutic prothrombin complex (PT)-level after 4.3 and the "high-dose" group after 3.3 days. Heparin was discontinued after 6.0 and 5.0 days respectively. There was no difference in significant hemorrhage between the groups, and no clinical signs of progression of the thrombosis. In the second part of the study another 40 patients were randomized, followed with coagulation factor II, VII, IX and X and with repeated venograms. A stable therapeutic PT-level was achieved after 4.4 ("low-dose") and 3.7 ("high-dose") days, and heparin was discontinued after 5.4 and 4.4 days respectively. There were no clinical hemorrhages, the activity of the coagulation factors had dropped to the same level in both groups at the time when heparin was discontinued and no thromboembolic complications occurred. Our oral anticoagulation regimen with heparin treatment for an average of 4.4-5 days seems safe and reduces in-patient costs.

The use of a commercial ELISA for assay of thrombin-antithrombin complexes in purified systems.

Inhibition of thrombin by antithrombin III (AT) results in the formation of stable thrombin-AT complexes (TAT). An enzyme-linked immunosorbent assay (ELISA) following the sandwich principle is available for the determination of TAT complexes in human plasma, however, this ELISA method could not be used in purified systems containing thrombin and AT. It has therefore been modified for use in purified systems and an excellent correlation was found between the disappearance of thrombin and AT and the recovery of TAT complexes. Addition of thrombin inhibitor hirudin and heparin inhibitor polybrene into the reacting thrombin-AT mixture did not interfere with the assay of TAT. It was found that the use of siliconised tubes was necessary for the conservation of the TAT complexes.