Luca Spiezia

X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua)

We report a case of juvenile thrombophilia associated with a substitution of leucine for arginine at position 338 (R338L) in the factor IX gene (factor IX-R338L). The level of the mutant factor IX protein in plasma was normal, but the clotting activity of factor IX from the proband was approximately eight times the normal level. In vitro, recombinant factor IX-R338L had a specific activity that was 5 to 10 times as high as that in the recombinant wild-type factor IX. The R338 substitution causes a gain-of-function mutation, resulting in factor IX that is hyperfunctional.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca(2+)-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency

Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 +/- 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 +/- 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 +/- 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding.

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism.

BACKGROUND Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited. OBJECTIVES To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF(+)MPs) in plasma may contribute to cancer-associated thrombosis. PATIENTS/METHODS EMPs, PMPs and TF(+)MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500). RESULTS Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/μL, respectively) than controls (299 ± 102 and 495 ± 241 MP/μL; p<0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF(+)MPs (927 ± 415 MPs/μL) than controls (204 ± 112 MPs/μL; p<0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF(+)MPs plasma levels (1019 ± 656 MPs/μL) than cancer patients without VTE (755 ± 391 MPs/μL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF(+)MPs and VTE in cancer patients. CONCLUSIONS Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.

The PAI-I gene 4G/5G Polymorphism and Deep Vein Thrombosis in Patients with Inherited Thrombophilia

Genetic and acquired factors may influence phenotypic expression of inherited thrombophilia. Hypofibrinolysis due to excess PAI-I can be found in patients with deep vein thrombosis (DVT) and 4G/5G polymorphism of the PAI-1 gene may modulate the inhibitors synthesis. In 149 patients with inherited thrombophilia, the possible thrombotic contribution of both 4G/5G polymorphism and PAI-1 plasma levels was evaluated. Sixty-seven patients with idiopathic DVT and 98 normal subjects were also studied. By comparison with controls, a significantly higher prevalence of 4G/4G genotype was seen in idiopathic DVT and in thrombophilia patients, although in this latter group the difference only remained significant in cases symptomatic for thrombosis (p=0.01). The 4G/4G genotype was associated with a greater risk of thrombosis both in symptomatic thrombophilia patients (OR 2.85, 95% CI 1.26-6.46) and in idiopathic DVT patients (OR 3.1, 95% CI 1.26-7.59). The greater frequency of 4G allele in symptomatic thrombophilia patients with respect to controls was statistically significant (p=0.04). Compared to healthy subjects, PAI-i:Ag levels were higher in symptomatic thrombophilia patients and related to the 4G/5G polymorphism, with significantly higher values in the 4G/4G carriers. In conclusion, PAI-1 4G/5G polymorphism may influence PAI-i expression and thrombotic risk in patients with inherited thrombophilia.

Whole blood coagulation assessment using rotation thrombelastogram thromboelastometry in patients with acute deep vein thrombosis

Common tests for the assessment of blood coagulation in the acute phase of deep vein thrombosis are of limited value for the evaluation of the associated hypercoagulability. The new rotation thromboelastometry by rotation thrombelastogram has the potential to provide information on whole blood clot formation and prothrombotic state in patients with acute deep vein thrombosis. Rotation thrombelastogram parameters were evaluated in whole blood of 30 patients with a first episode of acute deep vein thrombosis and 40 healthy controls. The effect of factor VIII and fibrinogen levels on rotation thrombelastogram assays was also assessed in the study population and in a model of blood supplemented by increasing amounts of fibrinogen. All assays performed were consistent with a remarkable hypercoagulable profile in deep vein thrombosis patients as compared with controls. In particular, maximum clot firmness and the area under curve values, which are expected to better correlate with the hypercoagulable state in the acute phase of deep vein thrombosis, were significantly higher in patients than in controls. As expected, fibrinogen was shown to be one of the main determinants of the hypercoagulability in rotation thrombelastogram assays. In a small subset of acute deep vein thrombosis patients, inherited thrombophilia had no influence on rotation thrombelastogram parameters. The new rotation thrombelastogram thromboelastometry is a useful tool to detect acute deep vein thrombosis-related hypercoagulability. Prospective studies are needed to define the potential applications of rotation thrombelastogram in the management of deep vein thrombosis patients.

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia.

Summary.  Background: Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. Objective: To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non‐carriers with comparable prothrombin levels. Patients/methods: Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. Results: Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non‐carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver‐synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non‐carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels.Conclusions: Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC‐resistant than individuals with comparable prothrombin levels because of other causes.

Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from families with mixed type I/III protein S deficiency

Background Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. Design and Methods Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). Results Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families. Conclusions Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

Upper extremity deep vein thrombosis

Upper extremities deep venous thrombosis (UEDVT) is a rare condition. According to the literature, approximately 4–10% of all cases of venous thrombosis may involve the subclavian, axillary or brachial veins. In the last few decades, the incidence of UEDVT has increased because of more frequent use of central venous catheters (CVCs) and cardiac pacemaker implantation. In addition, another common risk factor for UEDVT is cancer. UEDVT is classified as primary, approximately one-third of cases, which refers either to effort thrombosis or idiopathic UEDVT, or secondary, due to the presence of overt predisposing causes. The onset of UEDVT is usually characterized by arm swelling and pain, but may also be completely asymptomatic especially in patients with a long-term presence of a CVC. Ultrasonography represents a simple and accurate diagnostic tool to demonstrate the problem. UEDVT has major clinical consequences including pulmonary embolism, recurrences, post-thrombotic syndrome, and death. The role of thromboprophylaxis for those patients with a long-term CVC is still controversial. Unfractionated or low molecular weight heparin, followed by an oral anticoagulant are the most common treatments, with strategy of management similar to that of deep vein thrombosis of the leg. Thrombolysis/thrombectomy and surgical decompression are often successful, but less frequently used. Randomized controlled trials are warranted to clarify the optimal management of UEDVT, and to identify patients at the highest risk of recurrence who might benefit from long-term anticoagulation.

Plasma fibrinolytic capacity in renal transplant recipients: Effect of steroid-free immunosuppression therapy

Background. Cardiovascular disease is the most common cause of death among renal transplant recipients (RTRs). Impaired fibrinolytic capacity caused by an increase in plasminogen activator inhibitor type 1 (PAI-1) levels is involved in the onset of atherosclerosis and thrombotic complications. Long-term steroid treatment may induce arterial hypertension and metabolic and prothrombotic changes (including up-regulation of PAI-1 synthesis), which increase the cardiovascular risk. We evaluated plasma fibrinolytic behavior in two groups of RTRs treated with different immunosuppressive regimens. Methods. Twenty-seven RTRs were randomized to receive long-term (17 patients) or perioperative short-term (10 patients) steroids in addition to immunosuppression with cyclosporine A plus everolimus (Certican; Novartis, Basel, Switzerland) (7 patients) or FK506 plus mycophenolate mofetil (20 patients). In each patient, fibrinolytic capacity was studied with the 20-min venous occlusion test 1 and 6 months after transplantation. The following were assayed: euglobulin lysis time, tissue-type plasminogen activator antigen, and PAI-1 antigen and activity. Results. One month after transplantation, a severe impairment of fibrinolytic capacity, mainly caused by an increase in PAI-1 antigen and activity levels, was seen in patients with and without steroid treatment. Six months after transplantation, an improvement in fibrinolytic potential as the result of a decrease in PAI-1 levels was observed only in patients without steroid therapy. None of the steroid-treated patients demonstrated PAI-1 values correlating with body mass index, blood pressure, and metabolic parameters, thus confirming the effect of exogenous factors on PAI-1 expression. Moreover, all patients revealed a slight impairment of stimulated endothelial tissue-type plasminogen activator release, regardless of any steroid treatment, which was probably attributable to calcineurin inhibitor-induced endothelial dysfunction. Conclusions. Our study suggests that steroid-free immunosuppression is associated with a better fibrinolytic capacity in RTRs. This finding may contribute toward reducing the risk of cardiovascular events.