Inhibition of factor XI reduces thrombus formation in rabbit jugular vein under endothelial denudation and/or blood stasis

Introduction

In addition to acquired and inherited risk factors, the growth of venous thrombus under static conditions and endothelial injury play important roles in the development of deep venous thrombosis (DVT), for which risk factors include increased plasma levels of coagulation factor XI (FXI). The aim of this study is to understand the role of FXI in venous thrombus formation under conditions of endothelial denudation and/or blood stasis.

Materials and methods

The contribution of FXI to venous thrombus formation was investigated in a rabbit model and a flow chamber system. Thrombi were induced in the rabbit jugular veins by (1) endothelial denudation, (2) vessel ligation (blood stasis) or (3) by combined endothelial denudation and vessel ligation. Blood samples were perfused on immobilized type III collagen at a wall shear rate of 70/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. Prothrombin fragment 1 + 2 (F1 + 2) generation was also measured before and after perfusion.

Results

All thrombi induced in rabbit jugular veins were composed of platelets, fibrin and erythrocytes. Anti-FXI antibody significantly reduced ex vivo plasma thrombin generation initiated by ellagic acid but not by tissue factor, and in vivo thrombus formation under endothelial denudation and/or vessel ligation. The antibody significantly reduced surface areas covered by platelets and fibrin, as well as F1 + 2 generation at a wall shear rate of 70/s in flow chambers.

Conclusion

These results suggest that FXI contributes to venous thrombus growth under conditions of endothelial denudation and/or blood stasis, and that thrombin generation by FXI interaction promotes further platelet aggregation and fibrin formation at low shear rates.     

Lack of P-selectin glycoprotein ligand-1 protects mice from thrombosis after collagen/epinephrine challenge

Introduction

In thrombotic processes, during the association of leukocytes with platelets and endothelial cells, P-selectin glycoprotein ligand-1 (PSGL-1) binds to P-selectin, expressed on activated platelets and endothelial cells. Our aim was to establish the role of PSGL-1 in thrombus formation by evaluating the response to thrombotic stimuli in wild type and PSGL-1 knockout mice.

Materials and methods

Mice were challenged by tail vein injection of (i) 15 μg collagen plus 3 μg epinephrine (coll/epi) (ii) 7.5 μg collagen plus 1.5 μg epinephrine or (iii) saline. Retro-orbital blood samples were collected in ACD anticoagulaed tubes and platelet and leukocyte counts were measured. In addition, kidneys, liver, spleen and lungs were investigated for fibrin deposition by immunohistochemistry and Western-blotting. Frozen sections were analysed for double labeling for platelet and leukocyte presence.

Results

After coll/epi challenge, the number of platelets and leukocytes decreased significantly in both genotypes. Lower agonist concentration resulted in an attenuated platelet decrease in PSGL-1 knockout mice compared to the controls, however changes in leukocyte and neutrophil counts were not significantly different in the two strains. In knockout mice considerably less fibrin deposition has been observed in the lungs by Western-blotting and immunohistochemistry. After coll/epi challenge the lungs of the PSGL-1 knockout animals contained both platelets and leukocytes but less thrombi has been detected than in wild-type mice.

Conclusions

Our results indicate that the deficiency of PSGL-1 results in milder thrombocytopenia, less fibrin deposition and lower number of thrombosed blood vessels, suggesting that this molecule is essential for multicellular interactions during thrombus formation.     

Partial versus complete factor VIII inhibition in a mouse model of venous thrombosis

Introduction

Partial inhibition of Factor VIII (FVIII) may provide antithrombotic efficacy whilst avoiding excessive anticoagulation.

Materials and Methods

We studied the anticoagulant effects of a partial (TB-402) and a complete (BO2C11) FVIII-inhibiting monoclonal antibody (MAb) on FVIII, aPTT, thrombin generation and fibrin deposition in a flow chamber model. The antithrombotic efficacy of TB-402 and BO2C11 was compared in a mouse model of venous thrombosis.

Results

Both in vitro and ex vivo, the maximally achievable FVIII inhibition by TB-402 was about 25 to 30%. The degree of inhibition reached a plateau in vitro at 0.316 μg/mL and ex vivo after administering 0.1 mg/kg and higher doses. BO2C11 strongly inhibited FVIII:C, up to 91% at 100 μg/mL in vitro, and by 88% ex vivo 1 hour after administering 1 mg/kg to the mice.Whereas BO2C11 also markedly prolonged the aPTT and completely inhibited thrombin generation in vitro and ex vivo, the effect of TB-402 on the aPTT and on thrombin generation was limited. Similarly, in a dynamic flow chamber model, TB-402 and BO2C11 inhibited tissue factor-induced human fibrin deposition by 40% and 76%, respectively.In a mouse model of FeCl₃-induced venous thrombosis, TB-402 (1 mg/kg) inhibited thrombus formation to the same extent as BO2C11 (2 mg/kg) and enoxaparin (5 mg/kg), with a mean (± SD) occlusion time of 51 ± 13 minutes for TB-402, compared to 28 ± 6 minutes for the controls, 51 ± 13 minutes for BO2C11 and 55 ± 11 minutes for enoxaparin.

Conclusions

In this mouse model of venular thrombosis, partial FVIII inhibition yielded similar antithrombotic effects as nearly complete FVIII inhibition. These preclinical data are indicative of a therapeutic potential of partial FVIII inhibition in the management of venous thromboembolism.     

The effect of physiologically relevant dynamic shear stress on platelet and endothelial cell activation

Introduction

Blood flow induced shear stress plays an important role in platelet and endothelial cell functions. The goal of this study was to investigate the effect of physiologically relevant dynamic shear stress on platelet and endothelial cells.

Materials and Methods

Pulsatile shear stress waveforms mimicking the flow in a normal left coronary artery (0.1-1 Pa), at a 60% stenosis (0.2 - 6 Pa) and in the recirculation zone (0.01 - 0.5 Pa) behind a stenosis were used to stimulate platelets and endothelial cells in a cone and plate shearing device. Platelet activation was measured by CD62P expression and thrombogenicity. Meanwhile, endothelial cell activation and damage was measured by cell surface ICAM-1 and tissue factor expression using fluorescence microscopy. Endothelial tissue factor activity was measured using a commercial kit.

Results

Results showed that for platelets, a short exposure to elevated shear stress at the stenosis throat did not induce significant increase in platelet activation or thrombogenicity. While the low pulsatile shear stress had a potential for enhanced thrombosis. Both low and high pulsatile shear stress led to a significant increase in ICAM-1 expression on endothelial cell surface, but only low shear stress caused tissue factor over expression and enhanced tissue factor activity.

Conclusion

These results suggest that low pulsatile shear stress may be more atherogenic, compared to elevated shear stress induced by stenosis.     

The G534E-polymorphism of the gene encoding the Factor VII-activating protease is a risk factor for venous thrombosis and recurrent events

Introduction

A single nucleotide polymorphism of the factor VII activating protease (FSAP), FSAP Marburg I (rs7080536) has been identified as a risk factor for venous thrombosis, but its clinical role has so far been controversial in part due to small cohort sizes. The aim of the present case-control study was to elucidate the impact of the FSAP Marburg I polymorphism (FSAP-MI) on the development of venous thromboembolic disease (VTE) with other known sequence variations, including Factor V Leiden (rs6025) and Factor II G20210A (rs1799963).

Materials and Methods

The study included 891 patients (312 male and 579 female) with a history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) and 1283 healthy blood donors with no history of venous thromboembolic disease.

Results

We found that besides to the well-established aforementioned sequence variations of FV and Prothrombin, the FSAP Marburg I (FSAP-MI) polymorphism was significantly associated with the development of DVTs (1.65 (1.16-2.34) OR (95% CI)) and recurrent thromboembolic events (DVT and PE) (2.13 (1.35-3.36) OR (95% CI)). Comparing patients displaying one or more events FSAP-MI was still associated with the development of recurrent thromboembolic events (1.64 (1- 2.69) OR (95% CI)).

Conclusions

We conclude that FSAP Marburg-I genotyping may be used to determine the risk for thromboembolic disorders in patients with suspected thrombophilia and known DVT or PE.     

The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets

Introduction

Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet α-granule proteins such as VEGF and endostatin.

Materials and methods

Human isolated platelets were incubated with thrombin (0.5 U/ml), PAR1-activating peptide (AP) (0.4-30 μM) or PAR4-AP (1.5-300 μM) for up to 24 hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin.

Results

Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24 hours incubation of platelets.

Conclusions

PAI-1 and VEGF, but not endostatin, might be stored in the same α-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24 hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.     

Safety of low-dose subcutaneous enoxaparin for the prevention of venous thromboembolism after primary intracerebral haemorrhage

Background

The risks and benefits of low molecular weight heparins (LMWH) for the prevention of deep-vein thrombosis (DVT) and pulmonary embolism (PE) after intracerebral haemorrhage (ICH) have not been assessed. The few studies on this subject have revealed conflicting results.

Methods

We retrospectively evaluated whether subcutaneous enoxaparin (20 mg daily) reduced symptomatic venous complications or caused increased 3-month death rate. We included 407 patients who were admitted to a stroke unit and survived the first two days after onset of ICH. There were 232 patients who received anticoagulant treatment for the prevention of DVT and PE and 175 who did not.

Results

Despite the fact that the treated patients were in worse clinical condition at the start of the treatment, 3-month death rate was 19% among them compared to 21% among those not receiving anticoagulant therapy. Low-dose subcutaneous enoxaparin (20 mg once daily) induced a significant plasma anti-factor Xa activity 2-3 hours after administration (p = 0.018). Haematoma enlargements (33%) occurred in 9% and 7% of the treated and untreated patients, whereas symptomatic venous thromboembolic complications were observed in 3% and 2%, respectively.

Conclusions

We did not observe any increased mortality among ICH patients who survived the first 2 days after the onset of ICH and were thereafter treated with enoxaparin 20 mg daily relative to patients remaining untreated. A randomized trial of the effect of LMWH with a higher dose in prevention of venous thromboembolic complications would be indicated.     

The incidence of deep vein thrombosis in women undergoing cesarean delivery

Introduction

Venous thromboembolism (VTE) is one of the leading causes of maternal mortality in the United States. Cesarean delivery is a known risk factor. This study was to determine the incidence of deep vein thrombosis (DVT) post cesarean delivery.

Materials and Methods

This was a prospective cohort study where two patients having undergone cesarean delivery each day were randomly selected. A lower extremity compression ultrasound was performed prior to hospital discharge. If no DVT was detected, participants were asked to return for a second ultrasound two weeks postpartum. Participants were also telephone-interviewed at three months for reported VTE.

Results

Of the 194 patients who consented to study participation, only one participant developed DVT after cesarean delivery, giving an overall incidence of 0.5% (95% CI, 0.1 to 2.8%). There were no DVT identified on the second ultrasound nor VTE reported 3 months postpartum.

Conclusions

We found the DVT rate after cesarean delivery to be 0.5%.     

Formation of the venous thrombus after venous occlusion in the experimental mouse model of metabolic syndrome

Introduction

The metabolic syndrome is considered to be a risk factor for the venous thromboembolism (VTE) as well as arterial thrombosis. Although obesity, hyperglycemia and dyslipidemia are considered to be important triggering factors, it is difficult to evaluate the relationship between VTE and the metabolic syndrome in a clinical study. Furthermore the mechanism of venous thrombosis initiation still remains elusive.

Materials and Methods

20 min clamp of superior mesenteric vein was applied to 7 w, 16 w-old KK-A^y mouse and 16 w-old B6J mouse (n = 6 in each group), after de-clamp, the view of the mesenteric vein and intestinal submucosal venule were observed by the intravital microscopy.

Results

Massive thrombi formed in the mesenteric vein in 16 w-old KK-A^y mice, moderate thrombi formation was observed in 7 w-old KK-A^y mice, while very few thrombi were observed in B6J mice. The first event in submucosal venule after de-clamp was the adhesion of leukocytes to the endothelium. Subsequently, leukocytes assembled and platelets covered the leukocyte cluster. These leukocyte-platelet aggregates move from the venule to the vein and finally formed a venous thrombus.

Conclusion

Metabolic syndrome is a risk factor for venous thrombosis. Intravital microscopic examination revealed leukocyte and platelet recruitment to the venule in the early stages of venous thrombosis formation.     

Clinical probability assessment and D-dimer determination in patients with suspected deep vein thrombosis, a prospective multicenter management study

Objectives

To investigate the reliability of a combined strategy of clinical assessment score followed by a local D-dimer test to exclude deep vein thrombosis. For comparison D-dimer was analysed post hoc and batchwise at a coagulation laboratory.

Design

Prospective multicenter management study.

Setting

Seven hospitals in southern Sweden.

Subjects

357 patients with a suspected first episode of deep vein thrombosis (DVT) were prospectively recruited and pre-test probability score (Wells score) was estimated by the emergency physician. If categorized as low pre-test probability, D-dimer was analysed and if negative, DVT was considered to be ruled out. The primary outcome was recurrent venous thromboembolism (VTE) during 3 months of follow up.

Results

Prevalence of DVT was 23.5% (84/357). A low pre-test probability and a negative D-dimer result at inclusion was found in 31% (110/357) of the patients of whom one (0.9%, [95% CI 0.02-4.96]) had a VTE at follow up. Sensitivity, specificity, negative predictive value and negative likelihood ratio for our local D-dimer test in the low probability group were 85.7%, 74.5%, 98.2%, and 0,19 respectively compared to 85.6%, 67,6%, 97.9% and 0,23 using batchwise analysis at a coagulation laboratory.

Conclusion

Pre-test probability score and D-dimer safely rule out DVT in about 30% of outpatients with a suspected first episode of DVT. One out of 110 patients was diagnosed with DVT during follow up. No significant difference in diagnostic performance was seen between local D-dimer test and the post hoc batch analysis with the same reagent in the low probability group.     

Associations between high factor VIII and low free protein S levels with traditional arterial thrombotic risk factors and their risk on arterial thrombosis: Results from a retrospective family cohort study

Introduction

Whether high factor (F)VIII and low free protein S levels are risk factors for arterial thrombosis is unclarified.

Material and Methods

In a post-hoc analysis of a single-centre retrospective family cohort, we determined if these two proteins could increase the risk of arterial thrombosis. In total, 1399 relatives were analysed.

Results

Annual incidence in relatives with high FVIII levels was 0.29% (95%CI, 0.22-0.38) compared to 0.13% (95%CI, 0.09-0.19) in relatives with normal FVIII levels. In relatives with low free protein S levels, this risk was 0.26% (95%CI, 0.16-0.40), compared to 0.14% (95%CI, 0.10-0.20) in relatives with normal free protein S levels. Mean FVIII levels adjusted for age and sex were 11 IU/dL, 18 IU/dL, and 21 IU/dL higher in relatives with hypertension, diabetes mellitus, and obesity as compared to relatives without these arterial thrombotic risk factors. Moreover, a dose response relation between increasing FVIII and body mass index was found. None of these associations were shown for free protein S.

Conclusions

High FVIII and low free protein S levels seemed to be mild risk factors for arterial thrombosis. High FVIII levels were particularly observed in relatives with traditional arterial thrombotic risk factors. Free protein S levels were not influenced by these thrombotic risk factors. This assumes that low free protein S levels were genetically determined.     

Activated inflammatory cells participate in thrombus size through tissue factor and plasminogen activator inhibitor-1 in acute coronary syndrome: Immunohistochemical analysis

Introduction

Recent studies have suggested that circulating inflammatory cells augment the growth of thrombus in acute coronary syndrome (ACS). We therefore immunohistochemically analyzed thrombi in aspirates obtained from patients immediately after the onset of ACS.

Materials and Methods

Two hundred twenty samples were studied. Total thrombus area, white thrombus area, and red thrombus area were measured. As antibodies in immunohistochemical staining, myeloperoxidase (MPO), CD66b, CD68, p-selectin, tissue factor (TF) and PAI-1were employed respectively.

Results

The ratios of areas of red and white thrombi correlated with whole sample areas of enlarged thrombi (r = 0.48, p < 0.001). The immunohistochemical findings revealed granulocytes and macrophages aggregated around p-selectin-positive platelets that shared the boundary between white and red thrombi, a region where MPO and CD66b expression was abundant in neutrophils. The ratios (%) of MPO- and CD66b-positive cells significantly correlated with whole sample areas (r = 0.50; p < 0.001 and r = 0.49; p < 0.001, respectively). Neutrophils and macrophages within thrombi were positive for TF and PAI-1. Along the boundary between red and white thrombi, TF and PAI-1 positivity coincided with MPO-, CD66b- and CD68-positive cells. The ratios of cells positive for both TF and PAI-1 in this area significantly correlated with the whole sample area (r = 0.43, p < 0.001 and r = 0.60, p < 0.001, respectively).

Conclusions

These results suggested that enhanced activation of peripheral neutrophils together with increased TF and PAI-1 expression might comprise a considerable portion of thrombus enlargement.     

A new method measuring the interaction between von Willebrand factor and coagulation factor VIII

Introduction

There is a need for more reliable methods measuring the binding of coagulation factor VIII (FVIII) to von Willebrand factor (VWF) in plasma samples, for use in the clinical routine. We have developed such a method measuring FVIII binding in plasma, utilizing an ELISA system.

Materials and Methods

Microtiter plates were coated with a monoclonal antibody (ESH-8), reacting with the C2 domain in FVIII. Thereafter the wells were treated with recombinant FVIII (Kogenate Bayer®). After washing, diluted plasma samples were added and incubated for 1 h. Then HRP-conjugated antibodies against VWF were added and used for quantification of bound VWF.

Results

A strong signal to VWF concentration response was obtained. Plasma from patients with different types of von Willebrand disease gave frequently diminished responses. However, after correction for the VWF antigen levels, by calculation of FVIII binding/VWF antigen ratio, only the patients with known von Willebrand disease type 2 N (n = 4) had clearly abnormal results. The FVIII binding in 40 healthy individuals was determined as 1.08 ± 0.48 U/mL (SD). After correction for the VWF antigen levels the result was 0.94 ± 0.15. Thus, the SD declined substantially by this correction. The within-series CV and between-series CV were determined as 6.8 and 11.3%, respectively.

Conclusions

We have established a simple and reliable method to detect decreased binding of FVIII to von Willebrand factor in plasma samples. The method can conveniently be used to study large populations, as well as finding minor binding defects in patients.     

Decreased fibrin network permeability and impaired fibrinolysis in the acute and convalescent phase of ischemic stroke

Introduction

We investigated fibrin network permeability and fibrinolysis in the acute and convalescent phase of ischemic stroke.

Methods

20 patients with a mean age of 74 years were studied in the acute (day 1) and convalescent phase (day 60) of ischemic stroke. 23 healthy individuals (controls) were also investigated. Fibrin formation in the samples was triggered by addition of tissue factor (1 pmol/L) and washed frozen-thawed platelets obtained from a healthy donor. The permeability constant (K_s), which reflects fibrin network permeability, was then calculated from liquid flow measurements. A global assay newly developed in our group was also employed to determine the balance between fibrin formation (“Coagulation profile”; Cp) and fibrin degradation (“Fibrinolysis profile”; Fp) in the same samples. We also measured PAI-1 antigen and fibrinogen concentrations in plasma.

Results

As compared to controls, the stroke patients had lower Ks (lower fibrin network permeability) both on day 1 and on day 60 (p < 0.01 and p < 0.05, respectively). Fibrinolysis, assessed by Fp, was reduced on both day 1 and day 60 (p < 0.001, compared to controls), and PAI-1 concentrations were increased (p < 0.01 for both, compared to controls). Fibrin formation capacity in plasma (i.e. Cp) was increased in the acute phase (p < 0.05) but not in the convalescence, as compared to controls.

Conclusion

The combination of a proneness to form a tighter fibrin network and impaired fibrinolysis is a feature of ischemic stroke that is present in both the acute and convalescent phase of the disease.     

Hypercoagulability after trauma: Hemostatic changes and relationship to venous thromboembolism

Background

Major trauma induces a hypercoagulable state, which is frequently complicated by pathological thrombosis. However the sequential changes in coagulation markers and their relationship to clinical thrombosis have been poorly characterized.

Methods

We measured several markers of in vivo coagulation and fibrinolysis and their regulation serially for 2 weeks after multi-system trauma in a prospective cohort of patients who received no anticoagulant prophylaxis. Asymptomatic deep vein thrombosis (DVT) was assessed by routine bilateral venography between day 12 and 14. Clinically suspected DVT and pulmonary embolism (PE) were investigated in a standardized manner.

Results

Among the 135 cohort patients the overall venous thromboembolism (VTE) rate was 59%. Markers of thrombin generation were markedly increased within 24 hours of injury, remained persistently elevated for about 5 days and then decreased by day 14. No early compensatory increase in Tissue Factor Pathway Inhibitor (TFPI) or the complex of Factor Xa and TFPI (FXa-TFPI) was seen; FXa-TFPI remained depressed throughout the study. There was no inverse relationship demonstrated between markers of thrombin generation and thrombin regulation. Acquired APC resistance and hypofibrinolysis did not appear to be important contributors to hypercoagulability after trauma. None of the coagulation markers were independently predictive of VTE. Increasing age was the only significant, independent predictor of VTE.

Conclusion

Major trauma leads to significantly increased and persistent thrombin generation with disruption of its regulation. Coagulation markers do not appear to add independent predictive value in detecting VTE. Increasing age is the most important clinical predictor of VTE after trauma.     

Physical activity in patients with deep venous thrombosis: a systematic review

Objectives

We performed a systematic review to assess the benefits or risks of physical activity in patients with an acute or previous DVT of the leg.

Data sources

PubMed, EMBASE and Science Citation Index were searched without language restrictions up to July 2007. Bibliographies of retrieved articles and personal files were also searched.

Review methods

Randomized trials and prospective cohort studies that included patients with acute or previous DVT, described an exercise intervention or exercise exposure, and described any related clinical outcome were selected. Data were independently extracted by 2 investigators.

Results

Seven randomized trials and two prospective observational studies were included. Early exercise, compared with bed rest, was associated with a similar short-term risk of pulmonary embolism in patients with acute DVT and led to more rapid resolution of limb pain. In patients with acute DVT, a 6 month daily walking program led to similar degrees of vein recanalization and improvement in quality of life as controls. In patients with previous DVT, 30 min of vigorous treadmill exercise did not worsen venous symptoms and improved calf muscle flexibility; a 6 month exercise training program improved calf muscle strength and pump function; and high levels of physical activity at one month tended to be associated with reduced severity of postthrombotic symptoms during the subsequent 3 months.

Conclusions

Early walking exercise is safe in patients with acute DVT and may help to reduce acute symptoms. Exercise training does not increase leg symptoms acutely in patients with a previous DVT and may help to prevent or improve the postthrombotic syndrome.     

Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers

Introduction

Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population.

Materials and methods

The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers.A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P).

Results

We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities.

Conclusions

Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.     

Has the incidence of deep vein thrombosis in patients undergoing total hip/knee arthroplasty changed over time? A systematic review of randomized controlled trials

Background

There is a perception in the orthopaedic and thromboembolism community that the incidence of deep vein thrombosis (DVT) has decreased in patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA).

Objectives

To assess the incidence of DVT with warfarin thromboprophylaxis over time in patients undergoing elective TKA or THA.

Methods

The MEDLINE, EMBASE, and Cochrane Library databases were searched to October 2006, supplemented by a manual search of reference lists. Two reviewers independently extracted data on study characteristics, quality and the frequency of total, symptomatic and proximal DVT.

Results

Fourteen studies (4,423 patients) were included. Total and proximal DVT after TKA declined over time (r = − 0.75, p = 0.031; r = − 0.86, p = 0.007 respectively). Total and proximal DVT after THA did not change. The risk of developing DVT after TKA was significantly higher than after THA (OR 1.85, 95% CI 1.6 - 2.14; p < 0.0001). The risk of developing symptomatic DVT after THA was significantly higher than after TKA (OR 2.18, 95% CI 1.11 - 4.27; p = 0.012).

Conclusions

The incidence of DVT in patients undergoing elective TKA appears to have declined in patients receiving warfarin thromboprophylaxis.     

The role of P4-P3' residues flanking Arg336 in facilitating activated protein C-catalyzed cleavage and inactivation of factor VIIIa

Introduction

Activated protein C (APC) inactivates factor VIIIa (FVIIIa) through cleavages at Arg336 in the A1 subunit and Arg562 in the A2 subunit. Proteolysis at Arg336 occurs 25-fold faster than at Arg562. Replacing residues flanking Arg336 en bloc with the corresponding residues surrounding Arg562 markedly reduced the rate of cleavage at Arg336, indicating a role for these residues in the catalysis mechanism.

Materials and Methods

To assess the contributions of individual P4-P3’ residues flanking the Arg336 site to cleavage efficiency, point mutations were made based upon those flanking Arg562 of FVIIIa (Pro333Val, Gln334Asp, Leu335Gln, Met337Gly, Lys338Asn, Asn339Gln) and selected residues flanking Arg506 of FVa (Leu335Arg, and Lys338Ile). APC-catalyzed inactivation of the FVIII variants and cleavage of FVIIIa subunits were monitored by FXa generation assays and Western blotting.

Results

Specific activity values of the variants were 60-135% of the wild type (WT) value. APC-catalyzed rates of cleavage at Arg336 remained similar to WT for the Pro333Val and Lys338Ile variants and was modestly increased for the Asn339Gln variant; while rates were reduced ~ 2-3-fold for the Gln334Asp, Leu335Gln, Leu335Arg, and Lys338Asn variants, and 5-fold for the Met337Gly variant. Rates for cofactor inactivation paralleled cleavage at the A1 site. APC slowly cleaves Arg372 in FVIII, a site responsible for procofactor activation. Using FVIII as substrate for APC, the Met337Gly variant yielded significantly greater activation compared with WT FVIII.

Conclusions

These results show that individual P4-P3’ residues surrounding Arg336 are in general more favorable to cleavage than those surrounding the Arg562 site.     

Hydrocephalus caused by a venous varix

Introduction

Vascular malformations are uncommon causes of aqueductal obstruction. To our concern, only one case of hydrocephalus caused by a venous varix draining an unruptured arteriovenous malformation has been described in the literature.

Case report

We report the case of a 70-year-old woman presenting with progressive cognitive impairment, gait disturbance and urinary incontinence. Magnetic resonance imaging and magnetic resonance angiography revealed a large venous varix draining an unruptured arteriovenous malformation causing hydrocephalus.

Conclusion

Although uncommon, it is necessary to consider the presence of a vascular malformation in adults who presented with signs of aqueductal obstruction.