Mutations in the shutter region of antithrombin result in formation of disulfide‐linked dimers and severe venous thrombosis

Summary. Background: Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. Objectives: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. Patients and methods: We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparin‐affinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. Results: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non‐expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide‐linked dimer of mutant antithrombin whose properties are consistent with head‐to‐head insertion of the reactive loop. Conclusions: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.     

蛋白C基因新突变His134Asn所致Ⅰ型蛋白C缺乏症

目的：研究一家族性血栓病相关病因的表型及基因型。方法：先证者、其母、二兄分别在３５，１９，３３岁起无明显诱因患反复性下肢深静脉血栓（ＤＶＴ）。对其四代１３个家庭成员的抗凝血酶Ⅲ、蛋白Ｃ（ＰＣ）、蛋白Ｓ（ＰＳ）、纤溶酶原的抗原和活性及活化的ＰＣ抗性等进行检测。结果：该家族５个成员患有Ⅰ型杂合子ＰＣ缺乏症（ＰＣ抗原和活性降低５０％左右）。ＰＣ基因各外显子及外显子、内含子连接区聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）未发现明显的异常带；亚克隆后测序发现ＰＣ的第Ⅵ外显子３４４４Ｃ→Ａ导致１３４Ｈｉｓ→Ａｓｎ变异，这是国外尚未报道的新突变，被命名为ＰＣ长沙。此突变使限制性内切酶ＨｐｈⅠ位点丧失，用ＰＣＲ／ＨｐｈⅠ进行家系分析，证实了６个家系成员（包括５个ＰＣ缺乏者）具有同样的突变。结论：Ｈｉｓ１３４Ａｓｎ是此家族ＰＣ缺乏所致ＤＶＴ的密切相关病理基因型。     

Mild antithrombin deficiency and risk of recurrent venous thromboembolism: results from the MEGA follow‐up study

Essentials

• Mild antithrombin deficiency may increase the risk of recurrent venous thromboembolism (VTE).
• In a cohort study, we stratified patients with VTE to various cut‐off antithrombin levels.
• A 1.6–3.7‐fold increased risk of recurrent VTE was observed in the lowest antithrombin categories.
• Mild antithrombin deficiency (activity < 5th percentile of normal) increases recurrent VTE risk.

Summary

Background

Mild antithrombin deficiency (previously defined as antithrombin activity below 70% or 80%) has been associated with a 2.4–3.5‐fold increased risk of recurrent venous thromboembolism (VTE). This finding may have implications for duration of antithrombotic therapy in VTE patients with mild antithrombin deficiency.

Objectives

To externally validate whether mild antithrombin deficiency is a risk factor for recurrent VTE.

Methods

In a population‐based cohort study, patients with a first VTE (n = 2357) were stratified according to percentile cut‐off antithrombin levels (< 5th [< 87%], 5–10th [87–92%], > 10th percentile [> 92%]) and functional antithrombin levels (< 70%, 70–80%, > 80%).

Results

During a median follow‐up of 7.4 years, 361 recurrent events occurred (incidence rate, 2.5/100 patient‐years). We observed an increased risk of recurrent VTE in the lowest antithrombin activity category (< 5th percentile; < 87%) as compared with antithrombin activity that was > 10th percentile (> 92%), with an adjusted hazard ratio (HR) of 1.5 (95%CI, 1.0–2.3). When analyses were stratified to antithrombin cut‐off criteria of< 70% vs. patients with antithrombin activity > 80%, the adjusted HR for venous recurrence was 3.7 (95% CI, 1.4–9.9). Mild antithrombin deficiency was able to predict recurrent VTE over at least 8 years of follow‐up and the association remained present when the population was stratified to the presence or absence of thrombosis risk factors. Restriction analyses, where patients who used anticoagulation at time of blood draw and those who reported drinking ≥ 5 glasses alcohol daily were excluded, did not materially affect these outcomes.

Conclusion

This study confirms that mild antithrombin deficiency is a risk factor for recurrent VTE.

     

Fondaparinux treatment of acute heparin‐induced thrombocytopenia confirmed by the serotonin‐release assay: a 30‐month, 16‐patient case series

See also Greinacher A. Immunogenic but effective: the HIT‐fondaparinux brain puzzler. This issue, pp 2386–8; Goldfarb MJ, Blostein MD. Fondaparinux in acute heparin‐induced thrombocytopenia: a case series. This issue, pp 2501–3. Summary. Background: Fondaparinux is theoretically an attractive agent for the treatment of immune heparin‐induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet‐activating anti‐platelet factor 4/heparin antibodies. Although reports of the use of fondaparinux for this indication have thus far been favorable, the diagnosis of HIT in most cases was not based on definitive laboratory confirmation of heparin‐dependent, platelet‐activating antibodies. Objectives: To report thrombotic and major bleeding outcomes with fondaparinux in patients with a high likelihood of having acute HIT based on clinical features and a positive result in the confirmatory platelet serotonin‐release assay (SRA), a sensitive and specific test for platelet‐activating HIT antibodies. Methods/Patients: We reviewed consecutive eligible patients with SRA‐positive HIT (mean peak serotonin release, 91% [normal, < 20%]; mean IgG‐specific PF4/heparin enzyme immunoassay result, 2.53 optical density units [normal, < 0.45 units]) in one medical center over a 30‐month period who received fondaparinux for anticoagulation during acute HIT (platelet count, < 150 × 10⁹ L^?1). Where available, plasma samples were used to measure thrombin–antithrombin (TAT) complex levels. Results: Sixteen patients with SRA‐positive HIT received fondaparinux: 14 surgical (11 after cardiac surgery; three after vascular surgery) and two medical (acute stroke). Fifty‐six per cent of patients had HIT‐associated thrombosis at the time of diagnosis. No patient developed new, recurrent or progressive thrombosis; one patient developed a major bleed (calf hematoma). One patient judged to have irreversible tissue necrosis before receiving fondaparinux therapy ultimately required limb amputation. TAT complex levels were reduced within 24 h of starting fondaparinux, and 13 of 13 patients were successfully switched to warfarin. Conclusion: Fondaparinux shows promise for the treatment of patients with SRA‐positive acute HIT.     

The multi‐functional serpin,protein C inhibitor: beyond thrombosis and hemostasis

Summary. Protein C inhibitor (PCI) is a member of the serine protease inhibitor (serpin) family. PCI was initially found to be an inhibitor of activated protein C, and later shown to be a potent inhibitor of blood coagulation and fibrinolysis such as that mediated by urokinase type‐plasminogen activator. Therefore, the protein came to be known as plasminogen activator inhibitor‐3. It also inhibits proteases involved in fertilization. PCI is broadly conserved, and is found in human, rhesus monkey, cow, rabbit, rat, mouse and chicken. The human PCI gene is located on chromosome 14q32.1 in a cluster of genes encoding related serpins. Sp1‐ and AP2‐binding sites in the 5′‐flanking region act as promoter and enhancer, respectively, for its expression in the liver. PCI mRNA is expressed in many organs in primates, but only in the reproductive organs in rodents. Recent studies using transgenic mice expressing the human gene have suggested that PCI is also involved in regulation of lung remodeling, tissue regeneration, vascular permeability, proteolysis in the kidney and tumor cell invasion. A protease inhibitor‐independent activity of PCI, the prevention of anti‐angiogenesis and metastasis of tumor cells, has also been observed. Thus, PCI is a unique multi‐functional serpin playing diverse roles in the thrombosis and hemostasis in multiple organs and tissues of a variety of species.     

Prevalence and predictors for post‐thrombotic syndrome 3 to 16 years after pregnancy‐related venous thrombosis: a population‐based,cross‐sectional,case‐control study

Background:  The long‐term outcome of pregnancy‐related venous thrombosis (VT) is not known. Objectives:  To assess predictors and long‐term frequency of post‐thrombotic syndrome (PTS) after pregnancy‐related VT. Patients/Methods:  In 2006, 313 women with pregnancy‐related VT during 1990–2003 and 353 controls answered a comprehensive questionnaire that included self‐reported Villalta score as a measure of PTS. Cases were identified from 18 Norwegian hospitals using the Norwegian Patient Registry and the Medical Birth Registry of Norway. The latter was used to select as possible controls women who gave birth at the same time as a case. Thirty‐nine patients and four controls were excluded because of VT outside the lower limbs/lungs or missing Villalta scores. Two hundred and four patients had DVT in the lower limb and 70 had pulmonary embolism (PE). The control group comprised 349 women naive for VT at the time of the index pregnancy. Results:  Forty‐two per cent of cases with DVT in the lower limb, compared with 24% of cases with PE and 10% of controls, reported a Villalta score of ≥ 5. Severe PTS (Villalta score of ≥ 15) was reported among 7%, 4% and 1%. Proximal postnatal, but not antenatal, thrombosis was a strong predictor of PTS with an adjusted odds ratio of 6.3 (95% confidence interval, 2.0–19.8; P = 0.002). Daily smoking before the index pregnancy and age above 33 years at event were independent predictors for post‐thrombotic syndrome. Conclusions:  PTS is a common long‐term complication after pregnancy‐related DVT. Proximal postnatal thrombosis, smoking and higher age were independent predictors of the development of PTS.     

The association of prothrombin A19911G polymorphism with plasma prothrombin activity and venous thrombosis: results of the MEGA study, a large population-based case–control study

BACKGROUND: Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G carriers of the prothrombin A19911G polymorphism has not been extensively investigated. OBJECTIVES AND METHODS: We assessed prothrombin activity, FIIG20210A, and FIIA19911G polymorphisms in a large population-based case-control study, the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Four thousand three hundred and sixty-five consecutive patients with a first episode of deep vein thrombosis of the leg or pulmonary embolism were included. The control group (n = 4779) consisted of partners of patients or persons gathered using a random-digit dialing method. We studied the effect of FIIA19911G polymorphism on prothrombin activity and thrombosis risk, also in combination with factor V Leiden. RESULTS: Among FII20210-GG control subjects, FII19911-GG carriers had 7.1% [95% confidence interval (CI): 5.7-8.5] higher mean prothrombin activity than FII19911-AA carriers and the risk for GG carriers was 1.43-fold increased compared to AA carriers [odds ratio (OR) 1.43; 95% CI: 1.27-1.61]. Among FII20210-GA control carriers, the mean prothrombin activity in both FII19911-AA and -AG carriers was nearly equivalent [131.7% and 133.4%; mean difference (95% CI) = 1.7% (-7.2-10.7)]. Because of genetic linkage, FII19911-GG carriers were very rare on a FII20210-GA background, as only one FII20210A carrier had FII19911-GG. In FII20210-GA carriers, the OR increased from 3.05 (95% CI: 2.17-4.27) in subjects with FII19911-AA to 3.33 (2.28-4.85) in subjects with FII19911-AG, compared to those with FII20210-GG and FII19911-AA. CONCLUSIONS: The FIIA19911G polymorphism is associated with mildly elevated prothrombin activity and is a risk factor for venous thrombosis.     

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization

Summary.  Background : We previously described a model of laser-induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb-IIIa and P2Y₁₂ inhibition, whereas only deep injuries were sensitive to thrombin blockade. Objective : The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. Results : A superficial injury was found to detach the endothelial cells and expose a collagen III- and IV-rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)-collagen and GPIb–von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI-immunodepleted mice and still strongly inhibited in VWF^−/− mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. Conclusions : This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.     

Survival and functional outcome following endovascular thrombectomy for anterior circulation acute ischemic stroke caused by large vessel occlusion in Sweden 2017–2019–a nationwide,prospective, observational study

BackgroundEndovascular thrombectomy (EVT) is standard of care for anterior circulation acute ischemic stroke (AIS) caused by large vessel occlusion (LVO), but data on nationwide performance in routine healthcare are sparse. The study aims were to describe EVT patients with LVO AIS, analyze mortality and functional outcome, and compare results with randomized controlled trials (RCTs).MethodsData from the Riksstroke and the Swedish Endovascular Treatment of Acute Stroke Registry (RSEVAS) on pre-stroke independent patients, with LVO AIS in 2017–2019, defined as occlusion of the intracranial internal carotid artery, or the M1 or M2 segments of the middle cerebral artery, and groin puncture <6 h of onset, were compared to aggregated HERMES collaboration RCT data. We assessed 90-day survival and function, defined by the modified Rankin Scale. Specific analyzes were stratified by occlusion location.ResultsIn all, 1011/2560 of RSEVAS patients matched RCT inclusion criteria. Compared with RCT data, patients were older (73 vs. 68), fewer received intravenous thrombolysis (63.1% vs. 83%), and M2 occlusions were more common (24.5% vs. 8%). 90-day survival in RSEVAS was 85.3%, 42.8% achieved good outcome and 5% had symptomatic intracerebral hemorrhage (sICH). Corresponding outcomes in RCT data were 84.7% survival, 46% good outcome, and 4.4% sICH. Functional outcome was most favorable following M2 occlusions.ConclusionsEVT patients from our large real-world national dataset differed from RCT patients in several baseline factors including distribution of vascular occlusion site. However, the overall outcome of EVT in our Swedish cohort appeared to well match the pivotal trial findings.