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Acute thrombotic microangiopathies (TMAs) are characterized by excessive microvascular thrombosis and are associated with markers of neutrophil extracellular traps (NETs) in plasma. NETs are composed of DNA fibers and promote thrombus formation through the activation of platelets and clotting factors.

Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bβ and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGAhomozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality.

The Outpatient Bleeding Risk Index (OBRI) and the Kuijer, RIETE and Kearon scores are clinical prognostic scores for bleeding in patients receiving oral anticoagulants for venous thromboembolism (VTE). We prospectively compared the performance of these scores in elderly patients with VTE.

Summary. Background:The Geneva Prognostic Score (GPS), the Pulmonary Embolism Severity Index (PESI) and its simplified version (sPESI) are well‐known clinical prognostic scores for a pulmonary embolism (PE).

Background: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin‐induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti‐PF4/heparin‐antibodies may assist clinical decision‐making. Objectives: To evaluate the performance of rapid ID‐H/PF4‐PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID‐H/PF4‐polymer lots (polystyrene beads coated with heparin/PF4‐complexes). Patients/Methods: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin‐induced thrombocytopenia between January 2000 and October 2008. Anti‐PF4/heparin‐antibodies were assessed by ID‐H/PF4‐PaGIA, commercially available ELISAs and heparin‐induced platelet aggregation test. Results: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low‐titre positive) in nine instances (1%; 95%CI, 0.4–1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty‐seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none – 82%; 95%CI, 66–98%), depending on the employed ID‐H/PF4‐polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT‐patients. Conclusion: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID‐H/PF4‐polymer lots, thus avoiding false negative results.

Background: Over the last 4 years ADAMTS‐13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS‐13‐deficient plasma (arbitrarily set at 0%) into one normal‐pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed‐vs.‐expected ADAMTS‐13 levels assessed as r2ranged from 0.931 to 0.998. Between‐run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F‐values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS‐13 levels (the higher the F‐value, the better the capacity) ranged from 3965 to 137. Between‐method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS‐13 offer the best performance characteristics. Conclusions: New assays for ADAMTS‐13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

Background: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS‐13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. Methods: ADAMTS‐13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. Results: ADAMTS‐13 activity was significantly lower in patients than in healthy controls [median 60% (range 27–160%) vs. 110% (range 63–200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS‐13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide rs = 0.484, P < 0.001; creatinine and soluble thrombomodulin rs = 0.596, P < 0.001). Conclusions: VWF parameters are reciprocally correlated with ADAMTS‐13 activity in severe sepsis and septic shock but have no prognostic value regarding outcome.

This overview summarizes the history of thrombotic thrombocytopenic purpura (TTP) from its initial recognition in 1924 as a most often fatal disease to the discovery in 1997 of ADAMTS‐13 deficiency as a major risk factor for acute disease manifestation. The cloning of the metalloprotease, ADAMTS‐13, an essential regulator of the extremely adhesive unusually large von Willebrand factor (VWF) multimers secreted by endothelial cells, as well as ADAMTS‐13 structure and function are reviewed. The complex, initially devised assays for ADAMTS‐13 activity and the possible limitations of static in vitroassays are described. A new, simple assay using a recombinant 73‐amino acid VWF peptide as substrate will hopefully be useful. Hereditary TTP caused by homozygous or double heterozygous ADAMTS‐13 mutations and the nature of the mutations so far identified are discussed. Recognition of this condition by clinicians is of utmost importance, because it can be easily treated and – if untreated – frequently results in death. Acquired TTP is often but not always associated with severe, autoantibody‐mediated ADAMTS‐13 deficiency. The pathogenesis of cases without severe deficiency of the VWF‐cleaving protease remains unknown, affected patients cannot be distinguished clinically from those with severely decreased ADAMTS‐13 activity. Survivors of acute TTP, especially those with autoantibody‐induced ADAMTS‐13 deficiency, are at a high risk for relapse, as are patients with hereditary TTP. Patients with thrombotic microangiopathies (TMA) associated with hematopoietic stem cell transplantation, neo‐plasia and several drugs, usually have normal or only moderately reduced ADAMTS‐13 activity, with the exception of ticlopidine‐induced TMA. Diarrhea‐positive‐hemolytic uremic syndrome (D+ HUS), mainly occurring in children is due to enterohemorrhagic Escherichia coliinfection, and cases with atypical, D− HUS may be associated with factor H abnormalities. Treatment of acquired idiopathic TTP involves plasma exchange with fresh frozen plasma (FFP), and probably immunosuppression with corticosteroids is indicated. We believe that, at present, patients without severe acquired ADAMTS‐13 deficiency should be treated with plasma exchange as well, until better strategies become available. Constitutional TTP can be treated by simple FFP infusion that rapidly reverses acute disease and – given prophylactically every 2–3 weeks – prevents relapses. There remains a large research agenda to improve diagnosis of TMA, gain further insight into the pathophysiology of the various TMA and to improve and possibly tailor the management of affected patients.

Background: Accurate estimates of the incidence of thrombotic thrombocytopenic purpura (TTP) are important to assess the resources required for current treatments as well as to anticipate the need to develop new treatments. Previous estimates have been indirect and have not reported data on patients with ADAMTS‐13 deficiency. Objective: To determine the incidence of patients with TTP‐hemolytic uremic syndrome (HUS) in three categories: all patients with clinically suspected TTP‐HUS, patients with idiopathic TTP‐HUS, and patients with severe ADAMTS‐13 deficiency. Methods: Incidence rates were estimated from the Oklahoma TTP‐HUS Registry, analyzing all 206 consecutive patients from January 1, 1996 to June 30, 2004 who were treated with plasma exchange for their initial episode of clinically suspected TTP‐HUS. ADAMTS‐13 activity was measured in 186 (90%) of the 206 patients. Results: The age–sex–race standardized annual incidence rates were 11.29 × 106(95% CI: 9.70–12.88) for all patients with clinically suspected TTP‐HUS; 4.46 × 106(95% CI: 3.43–5.50) for patients with idiopathic TTP‐HUS; and 1.74 × 106(95% CI: 1.06–2.41) for patients with severe ADAMTS‐13 deficiency (<5% activity). In all three categories, the incidence rates were greater for women and for blacks. For patients with severe ADAMTS‐13 deficiency, the age–sex standardized incidence rate ratio of blacks to non‐blacks was 9.29 (95% CI: 4.33–19.93). Conclusions: Accurate incidence rate estimates for all patients with clinically suspected TTP‐HUS, idiopathic TTP‐HUS, and TTP associated with severe ADAMTS‐13 deficiency have been determined. The greater incidence among women and blacks is comparable with their increased risk for other autoimmune disorders.

A severely deficient ADAMTS-13 activity (<5%) is a key laboratory finding confirming the diagnosis of thrombotic thrombocytopenic purpura (TTP), whereas a mildly or moderately decreased activity is found in various other conditions. Laboratory tests for ADAMTS-13 activity must reliably identify a severe deficiency and detect inhibitory antibodies against ADAMTS-13. We carried out a multicenter comparison of different assays for ADAMTS-13 activity in plasma, including the quantitative immunoblotting of degraded von Willebrand factor (VWF) substrate, the residual collagen binding activity and ristocetin cofactor activity of degraded VWF, and an immunoradiometric assay. The main goal was to investigate whether all assays concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies. ADAMTS-13 activity was determined by five laboratories in 30 plasma samples of patients with hereditary and acquired TTP and other conditions. ADAMTS-13 activity values of the samples ranged from <3% to > 100%. Concerning the identification of a severe ADAMTS-13 deficiency, good interassay and interlaboratory agreement was observed with only one false-negative and two false-positive results by two laboratories using a collagen binding assay. For samples with normal or mildly to moderately reduced ADAMTS-13 activity, results were less concordant. There was good agreement for the detection of strong inhibitors. We conclude that all assays investigated are useful as a screening test in suspected TTP. Further assay improvement is needed, however.

d-Dimer and fibrinogen are elevated in many diseases presenting signs and symptoms similar to those seen in patients with pulmonary embolism (PE). We tested the hypothesis that patients with PE have lower fibrinogen and higher d-dimer values than patients in whom the diagnosis is suspected but safely excluded. One hundred and ninety-one consecutive patients with suspected acute PE (85 positive, 106 negative) were investigated with a diagnostic strategy including d-dimer, pretest probability, and helical computed tomography as first-line tests. In 38 of 40 patients with suspected PE and d-dimer <500 µg L−1, PE was excluded without further testing. During a 3-month follow-up, there was no clinical PE among these 38 and the 68 patients with a negative helical CT. In 151 patients with d-dimer >500 µg L−1, d-dimer, fibrinogen, and d-dimer/fibrinogen ratio (D/F ratio) were different in PE-positive compared with PE-negative patients [medians (and ranges) for d-dimer: 3793 (780 – 42 195) vs. 992 (621–6957) µg L−1, fibrinogen: 3.8 (0.4–6.2) vs. 4.7 (2.2–8.4) g L−1, and D/F ratio: 1.22 (0.15–85.45) 103vs. 0.25 (0.09–1.03) × 103; P < 0.0001, respectively). The true positive rate was almost twice as high using D/F ratio >1.04 × 103(49 of 85 patients; 57.6%) compared with d-dimer >7000 µg L−1(25 of 85 patients; 29.4%). Patients with acute PE have lower fibrinogen values than patients with suspected but excluded PE. D/F ratio >103is highly specific for the presence of acute PE, and causes a doubling of the diagnostic rate compared with d-dimer testing alone.

High molecular weight (HMW) and low molecular weight (LMW) forms of von Willebrand factor (vWF) were isolated from normal human plasma in the presence of protease inhibitors. HMW and LMW vWF preparations were subjected to reduction of interdimeric disulfide bridges under mild reducing conditions. Following sodium dodecyl sulfate electrophoresis in 3% agarose, the vWF bands were detected by immunoblotting with a polyclonal rabbit anti-vWF antiserum as well as with two monoclonal antibodies directed against epitopes located in the NH2-terminal (MAb 418) or in the COOH-terminal (MAb 9) region of the vWF subunit. Our results suggest that the slowest migrating band of the dimeric triplet set of LMW vWF represents an asymmetric structure composed of an intact subunit to which one NH2-terminal and one COOH-terminal fragment are linked by disulfide bridges. The intermediate band of the first triplet of LMW vWF strongly reacted with MAb 9 but not with MAb 418, indicating that it represents a dimer of COOH-terminal fragments. The fastest migrating band of the same triplet is apparently a dimer of the NH2-terminal fragments because it reacted with MAb 418 but not with MAb 9. Each next higher family of triplets seems to contain one more asymmetric fragment of dimeric size. These results are compatible with a model according to which LMW forms of vWF are derived from HMW vWF by proteolytic cleavage in the circulating blood.

Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of ‘coarse’ network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent ‘fine’ gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.

Objectives Dural puncture is regarded a safe procedure when contraindications are carefully excluded and has so far not been recognised as a risk factor for cerebral venous sinus thrombosis (CVST). Five patients are described with CVST after dural puncture in the presence of additional risk factors. Methods In four out of five patients complete investigations for thrombophilia were performed at least one month after withdrawal of oral anticoagulation. Results In three out of four patients tested, activated protein C (APC) resistance due to heterozygous coagulation factor V R506Q mutation (factor V Leiden) was found. One patient was using oral contraceptives as a circumstantial risk factor and three had had spinal anaesthesia for surgical procedures. Family history of venous thromboembolism was negative in all patients. Retrospective evaluation of 66 patients with CVST disclosed that dural puncture was the fourth most common risk factor (8%) possibly contributing to thrombosis. Conclusion Dural puncture may constitute an additional risk factor for CVST especially in patients with APC resistance or surgery. In such patients a thrombophilia screen is indicated.

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity toward the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase that was followed by a relatively rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, lima bean, and ovomucoid trypsin inhibitors did not. The Ki of the reversible inhibitor benzamidine for autoactivation (240 microM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors. This indicates that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis of the time course of activation showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated by kallikrein. The apparent second-order rate constant was 2.7 × 10(4) M-1 s-1 (pH 7.2, 50 microM sulfatides, ionic strength I = 0.06, at 37 degrees C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the second-order rate constant of the reaction at high salt concentrations. In support of the autoactivation mechanism it was found that increasing the amount of kallikrein initially present in the reaction mixture resulted in a significant reduction of the lag period and a rapid completion of the reaction while the second-order rate constant was not influenced. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein.

Summary 1. In primary hyperparathyroidism an increased bone turnover is seen, accompanied by osteitis fibrosa (=fibroosteoclasia, FO) in severe forms of the disease. Both types of bone reaction may be detected by microradioscopy of X-rays of the hand, extensive striation of metacarpal cortical bone indicating increased bone turnover and subperiosteal resorption of phalanges pointing to FO.

Summary Report is given on a 68-year-old man who suffered primarily from progressive weight loss and repeated episodes of fever and arthralgia. Later, liver dysfunction and renal insufficiency developed. Liver and kidney biopsics disclosed granulomatous hepatitis and nephritis. Because of the morphologic and clinical findings, the diagnosis of Boeck's disease was made. Shortly before death, diarrhea developed. Autopsy revealed a massive systemic involvement in Whipple's disease proven by light and electron microscopy and immunofluorescence. Tuberculoid and epitheloid cell granulomas and isolated giant cells were found in addition to the biopsy findings in skeleton muscles, the small intestine, lymphnodes and bronchi. At autopsy, the kidney showed chronic interstitial nephritis. The literature of kidney involvement in Whipple's disease is reviewed. This is the first case with granulomatous interstitial nephritis and chronic renal insufficiency in an inadequately treated Whipple's disease.