Rob Fijnheer

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet‐rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22°C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation‐dependent antigens (GMP 140 from the alpha granules and a 53‐kDa glycoprotein from the lysosomal granules) was measured. β‐thromboglobulin (β‐TG) release was also determined. Disc‐to‐sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p < 0.01), had a higher β‐TG release (p < 0.01), bound more MoAbs against GP IIb/IIIa (p < 0.01), and expressed more GMP 140 and 53‐kDa glycoprotein (p < 0.01) than did BC‐ derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP‐derived platelets are more activated than BC‐derived platelets. This is most likely a result of the pelleting that follows the second high‐speed centrifugation of the PRP.

Soluble Adhesion Molecules Reflect Endothelial Cell Activation in Ischemic Stroke and in Carotid Atherosclerosis

BACKGROUND AND PURPOSE Activation of endothelial cells and platelets plays an important role in the development of atherosclerosis and thrombotic disorders. Soluble adhesion molecules originating from these cells can be demonstrated in plasma. We hypothesized that elevated plasma concentrations of soluble P-selectin (sP-selectin), soluble intercellular adhesion mole-cule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-selectin) can reflect activation of endothelial cells and/or platelets in acute ischemic stroke and in previously symptomatic internal carotid artery stenosis. METHODS Plasma was sampled from patients within 2 days of acute ischemic stroke (n = 28), from patients with a previous (> 1 week) transient or persistent ischemic neurological deficit associated with stenosis of the internal carotid artery (n = 34), and from control patients without a history of vascular disease (n = 34). Concentrations of sP-selectin, sICAM-1, sVCAM-1, and sE-selectin were measured by means of an enzyme-linked immunosorbent assay. RESULTS Compared with control subjects, sP-selectin and sE-selectin were significantly elevated in the acute stage of ischemic stroke (P < .0001 and P = .001, respectively) as well as in previously symptomatic carotid stenosis (P < .0001 and P = .0007). sICAM-1 and sVCAM-1 were not increased. CONCLUSIONS The elevated levels of sE-selectin indicate that endothelial cell activation occurs both in the acute stage of ischemic stroke and in previously symptomatic carotid atherosclerosis. Increased sP-selectin concentrations reflect endothelial cell activation as well but may also be caused by platelet activation.

Fcγ receptor polymorphisms in systemic lupus erythematosus: Association with disease and in vivo clearance of immune complexes

OBJECTIVE Fc receptors for IgG (FcgammaR) play a prominent role in the clearance of immune complexes in systemic lupus erythematosus (SLE). Polymorphisms of FcgammaR have been proposed as genetic factors that influence susceptibility to SLE. We analyzed 3 functional FcgammaR polymorphisms in a strictly Caucasian population of SLE patients, and determined the influence of these polymorphisms on the clearance of immune complexes in vivo. METHODS Genomic DNA was isolated from 230 Caucasian patients with SLE and 154 controls. Amplification of FcgammaR-genomic regions in allotype-specific polymerase chain reactions was used to distinguish the genotypes. In addition, we analyzed the FcgammaR genotypes of 13 patients with SLE who participated in a study determining the half-life of IgG-coated erythrocytes in the blood. RESULTS We found a strong trend toward skewing of FcgammaRIIa, with an enrichment of the homozygous FcgammaRIIa-R/R131 genotype in patients compared with controls. We did not find a correlation between this genotype and the development of lupus nephritis. However, we established that the half-life of IgG-coated erythrocytes in the blood was prolonged in patients expressing the FcgammaRIIa-R/R131 genotype. The homozygous FcgammaRIIIa-F/F158 genotype was found more frequently in patients with arthritis and/or serositis. CONCLUSION In Caucasian populations, the R/H polymorphism of FcgammaRIIa is a minor determinant in susceptibility to SLE, whereas the V/F polymorphism of FcgammaRIIIa is associated with a set of disease manifestations. Notably, the R/H polymorphism of FcgammaRIIa affects the clearance of immune complexes in vivo, which may influence the course of a disease such as SLE.

Progressive vascular damage in hypertension is associated with increased levels of circulating P-selectin

Objective To assess whether increased shedding of adhesion molecules in plasma provides an index for endothelial damage in hypertension. Design and methods Three groups of hypertensive patients with increasing severity of vascular damage were studied: 20 essential hypertensives, 21 atherosclerotic, renovascular hypertensives and four malignant hypertensives. Twenty healthy subjects were included as a control group. Levels of P-selectin, E-selectin, intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor in venous blood were measured, using sandwich-type enzyme-linked immunosorbent assay. Results For essential hypertensives a trend for increased P-selectin and E-selectin values compared with those in controls was observed (159 ± 44 versus 132 ± 40 ng/ml, P = 0.062 and 40 ± 13 versus 34 ± 17 ng/ml, P = 0.055, respectively). P-selectin (210 ± 84 ng/ml, P = 0.0021) and E-selectin (42 ± 12 ng/ml, P = 0.012) levels in renovascular hypertensives were significantly higher than those in healthy controls. There were no significant increases in circulating levels of intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor either in essential hypertensives or in renovascular hypertensives. Marked increases in circulating levels of adhesion molecules and von Willebrand factor relative to those in controls were observed in malignant hypertensives (P-selectin 634 ± 332 versus 132 ± 40 ng/ml, P = 0.0004; vascular cell adhesion molecule 968 ± 187 versus 493 ± 139 ng/ml, P = 0.0004; and von Willebrand factor 259 ± 75 versus 130 ± 72 U/dl, P = 0.016). Conclusions Progression of vascular damage in essential, renovascular and malignant hypertension is associated with a rise in circulating levels of P-selectins and, to a lesser extent, E-selectins, whereas levels of intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor are elevated only in diseases associated with acute severe vascular damage, including malignant hypertension. Our data suggest that selectins may be useful as indicators of vascular damage in hypertension.

Homocysteine in inflammatory bowel disease: a risk factor for thromboembolic complications?

OBJECTIVE:Patients with inflammatory bowel disease (IBD) are at increased risk for thromboembolic events. Hyperhomocysteinemia, which is an established risk factor for arterial as well as venous thrombosis, may be more prevalent in IBD because of vitamin deficiencies.METHODS:In this retrospective study, we studied the concentrations of total homocysteine (tHcy), cobalamin, folate, and pyridoxine in 231 consecutive patients with IBD, of whom 16 patients had a history of venous thrombosis, and nine a history of arterial thrombosis. Age- and gender-matched healthy volunteers served as controls (n = 102).RESULTS:Homocysteine concentrations in patients were higher (12.3 μmol/L [range 4.6–51.3] vs 11.1 μmol/L [range 3.9–27.6], p = 0.001) and hyperhomocysteinemia tended to be more prevalent in patients than in the controls (11.1% vs 5%, p = 0.07). Folate, cobalamin, creatinine, and pyridoxine concentrations were correlated with tHcy. Folate deficiency was infrequently encountered in IBD patients (4.3%). The tHcy concentration in patients with a history of venous or arterial thrombosis was not higher than in patients without a history of thrombosis (12.7 μmol/L [range 4.6–40.1] and 15.2 μmol/L (range 10.5–26.8) vs 12.3 μmol/L [range 10.5–26.8], not significant). Hyperhomocysteinemia was found in 18.8% of the patients with venous thrombosis, 11.1% of the patients with arterial thrombosis, and 10.5% of the patients without thrombosis (not significant).CONCLUSIONS:Hyperhomocysteinemia is a common phenomenon in IBD and correlates with serum folate, cobalamin, creatinine, and pyridoxine concentrations. No correlation between tHcy and a history of venous or arterial thromboembolic complications is found. Hyperhomocysteinemia does not seem to be a major contributory factor in the development of venous or arterial thrombosis in IBD patients.

The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients with TTP, antibodies are present that inhibit the von Willebrand factor (VWF) processing activity of ADAMTS13. We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed to ADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 microg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti-ADAMTS13 antibodies, we prepared a series of recombinant fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2-8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine-rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodies.

Whole-body MRI, including diffusion-weighted imaging, for the initial staging of malignant lymphoma: comparison to computed tomography.

Purpose:To assess the value of whole-body magnetic resonance imaging (MRI), including diffusion-weighted imaging (DWI), for the initial staging of malignant lymphoma, compared with computed tomography (CT). Materials and Methods:Thirty-one consecutive patients with newly diagnosed malignant lymphoma prospectively underwent whole-body MRI (T1-weighted and short inversion time inversion recovery [n = 31], and DWI [n = 28]) and CT. Ann Arbor stages were assigned by 1 radiologist according to whole-body MRI findings, and by another radiologist according to CT findings. Differences in staging between whole-body MRI (without and with DWI) and CT were resolved using other (imaging) studies (including 18F-fluoro-2-deoxyglucose positron emission tomography and bone marrow biopsy) and follow-up studies as reference standard. Results:Staging results of whole-body MRI without DWI were equal to those of CT in 74% (23/31), higher in 26% (8/31), and lower in 0% (0/31) of patients, with correct/incorrect/unresolved overstaging relative to CT in 3, 2, and 2 patients, respectively, and incorrect staging of both modalities in 1 patient. Staging results of whole-body MRI with DWI were equal to those of CT in 75% (21/28), higher in 25% (7/28), and lower in 0% (0/28) of patients, with correct/incorrect overstaging relative to CT in 6 and 1 patient(s), respectively. Conclusion:Our results suggest that initial staging of malignant lymphoma using whole-body MRI (without DWI and with DWI) equals staging using CT in the majority of patients, whereas whole-body MRI never understaged relative to CT. Furthermore, whole-body MRI mostly correctly overstaged relative to CT, with a possible advantage of using DWI.

An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (k(cat)/K(m)) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF.

Extremely high levels of von Willebrand factor antigen and of procoagulant factor VIII found in multiple myeloma patients are associated with activity status but not with thalidomide treatment.

Summary.  Venous thromboembolism (VTE) is a major complication in patients with multiple myeloma (MM) during treatment with thalidomide combined with chemotherapy and/or dexamethasone. The pathophysiology is not clear. We performed a cross‐sectional study in 20 MM patients who were treated with thalidomide for refractory/relapsed disease. Seven patients (35%) experienced an episode of VTE. Plasma samples were analyzed for known risk factors for VTE and compared with those from 30 MM patients without thalidomide treatment. The patients groups differed in their baseline characteristics in activity status only. Extremely high levels of factor VIII‐coagulant activity (FVIII:C, mean 352%) and von Willebrand factor antigen (VWF‐Ag, 374%) were found in all patients using thalidomide. All other prothrombotic risk factors were normal. In patients with VTE, VWF‐Ag but not FVIII:C, levels were significantly higher as compared with patients without VTE. Patients without thalidomide treatment had significantly lower levels of both coagulation factors but the difference was only due to difference in activity status. High FVIII:C/VWF‐Ag levels are found in patients with active MM and this is probably a reflection of increased bone marrow angiogenesis in MM. These prothrombogenic circumstances could contribute to the high incidence of VTE during treatment with thalidomide in combination with dexamethasone/chemotherapy.

Newly Diagnosed Lymphoma: Initial Results With Whole-Body T1-Weighted, STIR, and Diffusion-Weighted MRI Compared With 18F-FDG PET/CT

OBJECTIVE The purpose of this study was to compare whole-body MRI including diffusion-weighted imaging (DWI) with (18)F-FDG PET/CT in the staging of newly diagnosed lymphoma. SUBJECTS AND METHODS Twenty-two consecutively registered patients with newly diagnosed lymphoma prospectively underwent whole-body MRI (22 with T1-weighted, STIR, and DWI sequences and 21 with T1-weighted and STIR sequences but not DWI) and FDG PET/CT. Whole-body MRI-DWI was independently evaluated by two blinded observers. Interobserver agreement was assessed, and whole-body MRI-DWI was compared with FDG PET/CT. RESULTS The kappa values for interobserver agreement on whole-body MRI-DWI for all nodal regions together and for all extranodal regions together were 0.676 and 0.452. The kappa values for agreement between whole-body MRI-DWI and FDG PET/CT for all nodal regions together and for all extranodal regions together were 0.597 and 0.507. Ann Arbor stage according to whole-body MRI-DWI findings was concordant with that of FDG PET/CT findings in 77% (17/22) of patients. Understaging and overstaging relative to the findings with FDG PET/CT occurred in 0% (0/22) and 23% (5/22) of cases. In the care of 9% (2/22) of patients, overstaging with whole-body MRI-DWI relative to staging with FDG PET/CT would have had therapeutic consequences. CONCLUSION Our early results indicate that overall interobserver agreement on whole-body MRI-DWI findings is moderate to good. Overall agreement between whole-body MRI-DWI and FDG PET/CT is moderate. In the care of patients with newly diagnosed lymphoma, staging with whole-body MRI-DWI does not result in underestimation of stage relative to the results with FDG PET/CT. In a minority of patients, reliance on whole-body MRI-DWI leads to clinically important overstaging relative to the results with FDG PET/CT. FDG PET/CT remains the reference standard for lymphoma staging until larger-scale studies show that use of whole-body MRI-DWI results in correct staging in this minority of cases.