Howard H.W. Chan

Selective d-Dimer Testing for Diagnosis of a First Suspected Episode of Deep Venous Thrombosis: A Randomized Trial

BACKGROUND D-Dimer testing is sensitive but not specific for diagnosing deep venous thrombosis (DVT). Changing the use of testing and the threshold level for a positive test result on the basis of risk for DVT might improve the tradeoff between sensitivity and specificity and reduce the need for testing. OBJECTIVE To determine whether using a selective D-dimer testing strategy based on clinical pretest probability (C-PTP) for DVT is safe and reduces diagnostic testing compared with using a single D-dimer threshold for all patients. DESIGN Randomized, multicenter, controlled trial. Patients were allocated using a central automated system. Ultrasonographers and study adjudicators but not other study personnel were blinded to trial allocation. (ClinicalTrials.gov: NCT00157677) SETTING 5 Canadian hospitals. PATIENTS Consecutive symptomatic patients with a first episode of suspected DVT. INTERVENTION Selective testing (n = 860), defined as D-dimer testing for outpatients with low or moderate C-PTP (DVT excluded at D-dimer levels <1.0 µg/mL [low C-PTP] or <0.5 µg/mL [moderate C-PTP]) and venous ultrasonography without D-dimer testing for outpatients with high C-PTP and inpatients, or uniform testing (n = 863), defined as D-dimer testing for all participants (DVT excluded at D-dimer levels <0.5 µg/mL). MEASUREMENTS The proportion of patients not diagnosed with DVT during initial testing who had symptomatic venous thromboembolism during 3-month follow-up and the proportion of patients undergoing D-dimer testing and ultrasonography. RESULTS The incidence of symptomatic venous thromboembolism at 3 months was 0.5% in both study groups (difference, 0.0 percentage point [95% CI, -0.8 to 0.8 percentage points]). Selective testing reduced the proportion of patients who required D-dimer testing by 21.8 percentage points (CI, 19.1 to 24.8 percentage points). It reduced the proportion who required ultrasonography by 7.6 percentage points (CI, 2.9 to 12.2 percentage points) overall and by 21.0 percentage points (CI, 14.2 to 27.6 percentage points) in outpatients with low C-PTP. LIMITATION Results may not be generalizable to all D-dimer assays or patients with previous DVT, study personnel were not blinded, and the trial was stopped prematurely. CONCLUSION A selective D-dimer testing strategy seems as safe as and more efficient than having everyone undergo D-dimer testing when diagnosing a first episode of suspected DVT. PRIMARY FUNDING SOURCE Heart and Stroke Foundation of Ontario.

The IgG subclasses of platelet‐associated autoantibodies directed against platelet glycoproteins IIb/IIIa in patients with idiopathic thrombocytopenic purpura

Summary. The majority of patients with idiopathic thrombocytopenic purpura (ITP) have antiplatelet autoantibodies that are most frequently directed against platelet glycoproteins IIb/IIIa or Ib/IX/V. However, there is some debate whether the immune response is oligoclonal or polyclonal in nature. We investigated the subclass distribution of anti‐IIb/IIIa IgG autoantibodies in 59 prospectively studied patients with ITP. We also tested patients with a variety of thrombocytopenic disorders (n = 31) and healthy controls (n = 30). Platelet lysates were tested for IgG anti‐IIb/IIIa autoantibodies, and the specific IgG subclass distribution was measured using antigen capture assays. All testing was done blinded to diagnosis and other assay results. After unblinding, we found that 43 of the 59 ITP patients had anti‐IIb/IIIa autoantibodies (sensitivity = 73%). Anti‐IIb/IIIa autoantibodies were also detected in five of the 31 non‐ITP patients, but in none of the 30 healthy controls (specificity = 91%). The IgG subclass assay was positive in 39 of the 43 ITP patients with anti‐IIb/IIIa antibodies (sensitivity = 92%) and in 12 samples that had no detectable anti‐IIb/IIIa antibodies including two ITP patients (specificity = 83%). The most common subclass in the ITP patient samples was IgG1 (77%), either alone (n = 14) or with other IgG subclass antibodies (n = 19). However, there were also patients with only IgG2 (n = 2), IgG3 (n = 3) or IgG4 (n = 3) antibodies. Our results are consistent with the hypothesis that ITP is a heterogeneous disorder and that some patients have evidence of oligoclonality, whereas other patients have polyclonal autoantibodies.

Combination immunosuppressant therapy for patients with chronic refractory immune thrombocytopenic purpura

Treatment options for patients with chronic refractory immune thrombocytopenic purpura (ITP) are limited. Because combination immunosuppressant therapy appeared to be effective in ITP and other disorders, we used this approach in patients with particularly severe and refractory ITP. In this retrospective, observational study, we determined the response (platelet count above 30 x 10(9)/L and doubling of baseline) among 19 refractory ITP patients. Treatment consisted of azathioprine, mycophenolate mofetil, and cyclosporine. The patients had failed a median of 6 prior treatments, including splenectomy (in all except 1). Of 19 patients, 14 (73.7%) achieved a response lasting a median of 24 months, after which time 8 (57.1%) relapsed. Of the 8 relapsing patients, 6 responded to additional treatments. Of the 14 patients who achieved an initial response, 2 (14.3%) remained in remission after eventually stopping all medications. Severe adverse events did not occur. Combination immunosuppressant therapy can produce a rise in the platelet count that is sometimes sustained in refractory ITP patients.

Reversal of New, Factor-specific Oral Anticoagulants by rFVIIa, Prothrombin Complex Concentrate and Activated Prothrombin Complex Concentrate: A Review of Animal and Human Studies

INTRODUCTION Recombinant activated factor VII (rFVIIa), prothrombin complex concentrate (PCC) and activated PCC (aPCC) are three non-specific haemostatic agents sometimes employed to reverse new, factor-specific oral anticoagulants. METHODS We conducted a review in the literature to compare the abilities of rFVIIa, PCC and aPCC to reverse factor-specific anticoagulants. MEDLINE and EMBASE databases were searched up to Oct 2013. RESULTS Eleven animal studies and two human trials met predefined inclusion criteria. To account for dosing variations of anticoagulants among studies, data were interpreted based on standards referenced from human trials at therapeutic doses. In animal studies, inconsistencies in the reversal abilities of rFVIIa, PCC and aPCC can be partly attributed to inter-species differences in the affinity among various clotting factors and tissue factors. Moreover, the differences in the affinity between species-specific clotting factors and anticoagulants that were initially designed to inhibit human factor may impose additional obstacles when comparing single factor rFVIIa with agents that contained multiple clotting factors. In the absence of a common clinical indication for the utilization of rFVIIa, PCC and aPCC, it is difficult, if not impossible, to establish an equivalent dose among these haemostatic agents when comparing their effectiveness in reversing factor-specific oral anticoagulants. Human trials were too few and sub-optimally designed to draw definite conclusions. CONCLUSION While preclinical studies may hint at a role for these haemostatic agents in reversing the anticoagulant effects of oral, factor-specific anticoagulants, existing trials offer inconclusive evidence to guide a clinical decision among individual agents with respect to potency and thrombosis risk. The mechanistic differences of these hemostatic agents in terms of their interactions with other coagulation factors impose major obstacles for the scientists using animal models to compare the efficacy of these reversal agents.

Carotid artery dissections: thrombosis of the false lumen.

Carotid artery dissections are the second leading cause of stroke in young adults. The hemostatic response to a dissection involves exposure of the subendothelium to the intravascular environment. Platelet activation/aggregation superimposed by secondary coagulation cascade activity attempts to heal the injury. Failure of the hemostatic response to heal the injury may lead to further rupture of the intimal and medial layers, which allows for the blood to penetrate these layers to create a false lumen. Continued hemorrhaging into the false lumen may result in dissection progression or obstruction of blood supply to the true lumen and downstream blood vessels. The effects of thrombosis in the true versus false lumen may lead to opposite consequences. True lumen clotting may lead to ischemic complications of downstream cerebral vasculature, whereas false lumen clotting may lead to dissection healing. Current information on clinical outcomes and degree of false lumen clotting in a carotid dissection model is limited, and most of the available information on this controversial topic has been inferred from aortic dissections. Therefore in this report we summarize the present state of knowledge of the pathophysiology, detailed hemostatic response to the injury, clinical presentation and treatment of carotid dissections. We also emphasize the need for future studies to investigate the degree of false lumen clotting on the clinical outcomes of carotid dissections.

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

Background: The interaction of heparin with fibrinogen compromises its anticoagulant activity. Results: Zn2+ promotes heparin binding to His-544–His-545 on the fibrinogen α-chain. Conclusion: We identified a novel Zn2+-dependent heparin binding site on fibrinogen. Significance: Platelet release of Zn2+ at sites of vascular injury may promote heparin binding to fibrinogen, thereby further attenuating the anticoagulant activity of heparin. The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn2+ in this interaction because Zn2+ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn2+ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn2+-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn2+-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn2+ is present. These results reveal the mechanism by which Zn2+ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.

Acute renal vein thrombosis, oral contraceptive use, and hyperhomocysteinemia

Oral contraceptive use and hyperhomocysteinemia are considered to be relatively weak risk factors for venous thromboembolism. We report a case of acute renal vein thrombosis, a rare and aggressive form of thromboembolism, that occurred in a 21-year-old woman taking oral contraceptives, who was subsequently found to have marked hyperhomocysteinemia. This case suggests that the oral contraceptive and hyperhomocysteinemia may interact in a synergistic manner in the pathogenesis of thrombosis. In oral contraceptive users who develop venous thrombosis in the absence of other risk factors, clinicians should consider investigations for an underlying prothrombotic biochemical disorder.

Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin–heparin complex

The role of red blood cells (RBCs) in coagulation is not well understood. Overt exposure of phosphatidylserine on surfaces of RBCs provide docking sites for formation of the prothrombinase complex, which further aids in amplification of coagulation leading to subsequent thrombosis. No studies to date have evaluated heparin inhibition of the RBC-prothrombinase system. Therefore, this study examines the ability of heparin and a covalent antithrombin-heparin complex (ATH) to inhibit the RBC-prothrombinase system. Discontinuous inhibition assays were performed to obtain k₂ values for inhibition of free or prothrombinase-bound Xa by antithrombin and unfractionated heparin (AT + UFH) versus ATH. In addition, components of the complex (prothrombin, RBCs or Va) were excluded prior to reaction with inhibitors to investigate potential mechanisms involved. Inhibition of thrombin generation, fibrinogen conversion and plasma clotting by the RBC-prothrombinase system was also examined. Protection of Xa was observed for AT + UFH and not for ATH reactions. Inhibition rates for ATH were significantly faster when compared with AT + UFH results. The greatest impact on Xa inhibition was observed from factor Va omission for both inhibitors. ATH inhibited thrombin generation, fibrinogen conversion and plasma clotting better compared with AT + UFH. This study determined potential control of coagulation contributed by RBCs. Moreover, greater control of coagulation is achieved by covalently linking heparin to AT.

Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets

Factor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ~4-fold faster than AT+UFH. Relative to intact prothrombinase, ratesfor FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.

Coagulopathy in a patient with nephrotic syndrome

A 55-year-old Caucasian woman presented with a 5week history of increasing lower limb edema associated with a two-pound daily weight gain. She was feeling well otherwise; she denied shortness of breath, chest discomfort, recurrent headaches, myalgia, arthlagia, or constitutional symptoms. She had respiratory infection 4 months before, but the infection has resolved. The patient presented with peripheral edema. The differential diagnosis includes right-sided heart failure, liver cirrhosis, nephrotic syndrome, drug-induced edema, and lymphoedema. Her preceding upper respiratory tract infection suggests postinfectious cardiac or renal pathology. The patient did not have any prior bleeding episode. The medical history was significant for Graves’ disease treated with radioactive iodine ablation. The only medication was daily thyroid supplementation. She was married and had two uncomplicated pregnancies. She was a lifelong nonsmoker. She denied any alcohol intake or illicit drug use. She was a pharmacist without any known exposure to toxic chemicals. On examination her vital signs were normal. She was not in any distress. Examination of her legs revealed bilateral pitting peripheral edema up to the knee level. There was no jaundice, lymphadenopathy, or bleeding sign. Her cardiac examination was normal and her jugular venous pressure (JVP) was not elevated. Respiratory examination was normal with no crackles on auscultation. Abdominal examination revealed no palpable hepatosplenomegaly or ascites. The fact that this patient was not on any medication other than long term thyroid supplement makes druginduced edema unlikely. The finding of normal JVP on clinical examination is against the diagnosis of heart failure. Lymphedema is typically nonpitting. Thus, the most likely differential diagnosis includes nephrotic syndrome and early liver cirrhosis. Patient’s hemoglobin level was 19.0 g/dL (normal 11.5– 16.5 g/L), platelet count 305,000/mm (normal 150,000– 400,000/mm). The blood urea nitrogen was 14.4 mg/dL (normal 7.0–18.0 mg/dL), creatinine 0.93 mg/dL (normal <1.5 mg/dL), albumin 2.0 g/dL (normal 3.5–5.5 g/dL), cholesterol 587 mg/dL (normal <200 mg/dL), and triglyceride 447.1 mg/dL (normal <160 mg/dL). The results of liver function tests were normal. Urinalysis revealed 11 blood, 31 protein, and granular casts. Twenty-four hour urine protein excretion was 15,000 mg/d. The combination of heavy proteinuria, minimal hematuria, hypoalbuminemia, and hypercholesterolemia suggests nephrotic syndrome. Nephrotic syndrome may be associated with primary kidney disease, such as minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Systemic diseases including diabetes mellitus, systemic lupus erythematosus, amyloidosis, or Fabry’s disease also cause nephrotic syndrome. International normalized ratio (INR) was increased to 2.6 and activated partial thromboplastin time (aPTT) was prolonged to 68 s but thrombin clotting time was normal. Clauss fibrinogen level was slightly elevated at 480 mg/dL (normal range 200–400 mg/dL). D-dimer level was not elevated. In general, patients with nephrotic syndrome are in a hypercoagulable state because of urinary loss of antithrombin III and enhanced platelet reactivity, but this patient’s coagulation tests were paradoxically prolonged although there was no bleeding symptom. Simultaneous prolongation of both INR and aPTT suggests common coagulation pathway abnormalities like liver dysfunction, vitamin K deficiency, disseminated intravascular coagulopathy (DIC), primary fibrinolysis or post-thrombolytic therapy. However, the normal thrombin clotting time and borderline high fibrinogen level make abnormalities in the conversion pathway of fibrinogen to fibrin less likely. Normal fibrinogen and D-dimer level are atypical for DIC. A deficiency of multiple coagulation factors in both intrinsic and extrinsic coagulation pathways commonly occurs in vitamin K deficiency or hepatic dysfunction. However, 48 hr after administration of 5 mg oral vitamin K, the coagulation indices remained prolonged with an INR at 2.6 and aPTT at 63 s. To further define the causes of coagulopathy, clotting factor levels were assayed individually. Factor II level was high at 166%, factor VII 179%, but factor VIII:C was low at 7%, factor X 3%, whereas factor XII level was borderline low at 47%. (Normal range of these clotting factors was 50–150%). Although acquired factor XII deficiency has been reported in patients with nephrotic syndrome [1], deficiency of multiple coagulation factors suggests acquired conditions, either due to insufficient production or excessive consumption. Coagulation factors II, V, VII, IX, X, XI, and XII are synthesized in the liver, and in addition factor VIII also