Coagulation, fibrinolysis and platelet P-selectin expression in peripheral vascular disease.

OBJECTIVES: to examine coagulation, fibrinolysis, and platelet activity in patients with peripheral vascular disease (PVD). DESIGN: fifty consecutive PVD patients and 50 healthy volunteers. (Prospective comparative study.) MATERIALS AND METHODS: P-selectin expression in non-fixed, whole blood was measured flow cytometrically on non-stimulated and ADP- and TRAP-6-stimulated samples. Plasma fibrinogen, von Willebrand factor (vWF), tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 were determined using standard techniques. Disease severity was stratified on the basis of the ankle-brachial pressure index (ABPI) and the angiographic data were assessed using the Bollinger score. RESULTS: coagulation and fibrinolysis parameters as well as the P-selectin expression on both stimulated and non-stimulated platelets were significantly increased in patients vs controls (all p<0.01). The respective sensitivity and specificity were as follows: P-selectin expression (81%, 94%), vWF (72%, 86%), fibrinogen (64%, 98%), PAI-1 (44%, 90%), tPA (15%, 100%). P-selectin expression on TRAP-6-stimulated MP correlated with disease severity (r=0.40, p<0.01). CONCLUSIONS: these findings support the concept of ongoing thrombogenesis in the subclinical progression of PVD and demonstrate the high diagnostic sensitivity of flow cytometric analysis of platelet activation.

The cell function analyser (CFA) - a physiological in vitro vascular model and potential alternative to animal experiments.

Cell-vessel wall interactions (adhesion, emigration) and cell-cell cohesion (aggregation) have been assessed primarily in animal experiments. The cell function analyser (CFA) is an in vitro vascular model, in which the three components of Virchow's triad are present in a highly standardised and variable form. The CFA permits visual and quantitative analysis of cellular adhesion, emigration and aggregation under physiologically relevant flow conditions (i.e. to the arteries and to the microcirculation). Although the method does not entail the use of a living animal or of animal tissue, as is true for animal experiments, with the CFA specimen fixation and histomorphological analysis after the experiment is possible. The efficacy of the method for platelet function testing has been verified by numerous clinical studies. The wide variability of test parameters make CFA suitable for in vitro analysis of other cell-vessel-wall-mediated processes, such as inflammation, wound healing and tumour metastasis. We present: 1) a description of the CFA method and underlying hemodynamic principles, 2) a review of clinical and experimental results with platelets and, 3) the first results of convective flow-mediated leukocyte-endothelial interactions. The CFA provides an in vitro alternative to animal experiments, can be classified as a replacement method and possesses an analysis spectrum that will greatly reduce the overall need for the previous.

Mechanisms underlying increased platelet reactivity in patients with peripheral arterial disease. Preliminary results.

BACKGROUND: In peripheral arterial disease (PAD) atherosclerosis is disseminated and thrombosis risk is high. We have not only shown the platelets of PAD patients to by hyperreactive and aspirin resistant, but have recently verified them to be hypersensitive to heparin as well. In the present study we have begun to clarify the mechanisms underlying these regularly observed clinical findings. METHODS: Platelet function was tested with conventional, ADP-primed aggregation and with stagnation point flow adhesio-aggregometry (SPAA). SPAA permits real time, quantitative assessment of platelet adhesion and aggregation under biologically relevant flow conditions. The platelets from a female patient with congenital afibrinogenemia, were analyzed before and after intravenous fibrinogen substitution. In 14 PAD patients and 14 controls, platelet reactivity was assessed before and after incubation with the two platelet membrane glycoprotein (GP)-IIb/IIIa inhibitors Ro 43-8857 and 7E3. Lastly, experiments were performed before and after addition of plasma aliquots stemming from 4 PAD patients to platelet rich plasma and to solutions of gel-filtered platelets (GFP) stemming from 4 healthy volunteers. Before fibrinogen substitution, the platelets of the afibrinogenemic patient were unable to adhere in the SPAA-system and maximal ADP-primed aggregability was below 10%. After substitution, normal platelet adhesion was measured and primed aggregation increased three-fold. Mean baseline adhesion in the patient collective was twice that compared to controls (p<0.001). SPAA-measured, spontaneous aggregation was observed in ten patients and in none of the controls (p<0.001). RESULTS: Both SPAA-measured and primed aggregation were abolished at the lowest substrate concentrations (0.1 microM Ro 43-8857, 1 microg/ml 7E3). At these concentrations adhesion was reduced by 65% and 40% (respectively) in patients, and by 55% and 25% in controls. Total abolition of adhesion in both groups was seen with 0.5 microM Ro 43-8857 and 10 microg/ml 7E3. Platelet response to inhibitory agent was similar in patients and controls, as were the differences in dose-response between aggregation and adhesion. Upon addition of patient plasma to volunteer PRP, the platelets of all 4 healthy individuals aggregated spontaneously and the mean adhesivity in the group rose three-fold. The overall ability of the GFP to adhere when re-added to their own plasma was decreased, whereas, in the presence of patient plasma, adhesion increased significantly. CONCLUSIONS: On the basis of these findings we conclude that: 1) SPAA measures and quantifies platelet interactions with both fluid-phase (aggregation) and immobilized (adhesion) fibrinogen, 2) these reactions are mediated by the GP, IIb/IIIa receptor complex 3) the binding affinity, metabolic pathways and signal transduction underlying platelet adhesion differ from those involved in aggregation (possibly reflecting their varying roles in hemostasis), 4) the functionally normal platelets of patients with PAD are primed in vivo by a circulating plasma constituent, which leads to enhanced recruitment of activated GP, IIb/IIIa onto the platelet surface and, thereby, to an overall increase in reactivity.

Computational analysis of platelet adhesion and aggregation under stagnation point flow conditions.

The clinical relevance of platelet function assessment with stagnation point flow adhesio-aggregometry (SPAA) has been verified. Quantitative analysis of platelet adhesion and aggregation is possible by means of mathematical analysis of the dark-field, light intensity curves (growth curves) obtained during the SPAA experiment. We present a computational procedure for evaluating these curves, which was necessitated by, and is based on, actual clinical application. A qualitative growth curve classification, corresponding to a basic and distinct pattern of platelet deposition and characteristic of a regularly occurring clinical state is also presented.

Spreading of platelets: a morphological marker for platelet reactivity in peripheral arterial occlusive disease.

Platelet-surface contact is the first step in thrombus formation. Platelet spreading makes this initial contact irreversible. On the other hand plasma lipids and fibrinogen have been described to activate platelets or promote adhesion. We therefore investigated whether platelet spreading under stagnation-point flow conditions correlated with plasma concentrations of cardiovascular risk factors such as fibrinogen and high density lipoprotein (HDL)-cholesterol. Platelet rich plasma (PRP) from patients with peripheral arterial occlusive disease and healthy controls was examined by means of the Stagnation-Point Flow Adhesio- Aggregometer (SPAA). The SPAA comprises a microscopic setup with a flow chamber that permits direct observation and quantitation of platelet deposition onto standardized surfaces. After the flow experiments the deposited platelets were analyzed morphometrically for the degree of spreading expressed as inverse circularity (1/C). 1/C was correlated over 2 X 2 tables of fibrinogen combined with plasma levels of HDL-cholesterol, each of which was divided into a low and high value group. The patient and control group differed significantly with regard to 1/C, i.e. patient platelets demonstrated more adhesive platelets with a more extensive degree of spreading. 1/C was inversely correlated with HDL-cholesterol and showed significant differences between the patient and the control group. Increased 1/C values were found when associated with high fibrinogen levels and simultaneously with low HDL-cholesterol concentrations. Platelet spreading shows a correlation with increased levels of independent plasmatic risk factors for thrombosis in PAOD patients. Obtained during stagnation-point flow, spreading seems to be a morphological marker for platelet hyperreactivity.

Increased platelet and coagulatory activity in peripheral atherosclerosis flow mediated platelet function is a sensitive and specific disease indicator.

The importance of flow in both athero- and thrombogenesis is well established. In peripheral arterial disease (PAD) atherosclerosis is disseminated, thrombosis risk high and systemic hemostatic derangement believed contributory. We studied platelet and coagulatory activity in PAD patients, thereby evaluating the clinical efficacy of Stagnation Point Flow Adhesio-Aggregometry (SPAA). SPAA provides real-time quantitative assessment of platelet adhesion and aggregation under convective, low-shear flow conditions. 62 nondiabetic PAD patients and 66 healthy volunteers were examined, whereby SPAA and conventional aggregometry were performed and circulating fibrinogen, fibrin monomer (FM), the fibrin degradation product D-Dimer and thrombin-antithrombin-complex (TAT) assessed. Conventional aggregometry detected no differences between patients and controls. SPAA-measured platelet function (p < 0.001), fibrinogen (p < 0.001), FM (p < 0.001), TAT (p < 0.02) and D-Dimer (p < 0.001) were significantly increased in patients and not effected by aspirin therapy. Respective sensitivity and specificity in detecting PAD was as follows: SPAA (96%/95%), fibrinogen (36%/92%), FM (46%/88%), TAT (40%/73%), D-Dimer (75%/80%). Increased platelet and coagulatory activity was verified in PAD, whereby flow-mediated platelet adhesion and aggregation proved the most sensitive and specific parameter. These findings indicate the usefulness of SPAA for delineating platelet-related disease mechanisms and evaluating therapeutic strategies to prevent platelet activation.

Platelets of patients with peripheral arterial disease demonstrate a hypersensitive response to heparin.

We sought to verify earlier reports of increased platelet reactivity in patients with peripheral arterial disease (PAD) during perioperative heparin administration, and to test the hypothesis of platelet hypersensitivity to heparin in these patients. Before and after incubation of platelet rich plasma with unfractionated (UH), low molecular weight heparin (LMWH), and a low molecular weight heparinoid, real-time quantitative assessment of platelet function was performed by stagnation point flow adhesio-aggregometry (SPAA) in 21 patients with PAD and 14 healthy volunteers. With SPAA the occurrence of spontaneous aggregation is pathological. In the 15 patients requiring operation, platelet function and count were measured at regular intervals. To detect heparin dependent antibodies, the heparin induced platelet activation assay (HIPA) was performed preoperatively and after 10 days of heparin therapy. Mean baseline platelet adhesion in patients was double that observed in controls (p < 0.001). Spontaneous aggregation was seen in 9 (43%) patients and no controls (p < 0.001). In controls heparinoid reduced, whereas UH and LMWH slightly increased adhesion. Spontaneous aggregation was observed once with UH. Platelets from patients showed significantly enhanced adhesiveness and aggregability (p < 0.05) with UH and LMWH when compared to controls. Effects with the heparinoid were less pronounced and nonsignificant. In patients requiring operation, postoperative increases in platelet function and reductions in count were significant (p < 0.001). Ten (67%) experienced a fall in platelet count of > 50%. Preoperatively the HIPA assay showed no evidence of antibodies, whereas after heparin administration antibodies were verified in 4 (32%) patients and could not be ruled out in 6 (40%). Three developed postoperative thrombosis, in one case fatal. A hypersensitive in vitro and in vivo platelet response to heparin was verified in patients with PAD and a large number developed the immunological type of heparin-associated thrombocytopenia. Our findings suggest that a thrombin antagonist which does not interact with platelets may give the best perioperative protection in these patients.

Increased platelet and coagulatory activity indicate ongoing thrombogenesis in peripheral arterial disease.

In peripheral arterial disease (PAD) risk of thrombosis is high and systemic haemostatic derangement thought contributory. We investigated platelet and coagulatory activity in patients with PAD and sought to find the best disease indicator. Stagnation point flow adhesion-aggregometry (SPAA) enables real-time quantitative assessment of platelet adhesion and aggregation under well-defined flow conditions. SPAA and agonist-induced aggregometry (Born method) were performed and concentrations of fibrinogen, fibrin monomer (FM), D-dimer, and thrombin-antithrombin complex (TAT) measured in 92 PAD patients and 70 healthy volunteers. Agonist-induced aggregometry detected no differences between patients and controls. SPAA-measured platelet adhesion and spontaneous aggregation (p < 0.001), and concentrations of fibrinogen (p < 0.001), FM (p < 0.001), TAT (p < 0.02) and D-dimer (p < 0.001) were all significantly increased in patients. Neither platelet function nor coagulatory activity was altered in patients receiving aspirin. Sensitivity and specificity in detecting PAD were as follows: SPAA (95%, 93%), fibrinogen (36%, 91%), FM (48%, 84%), TAT (36%, 78%), D-dimer (73%, 80%). Our findings support the concept of ongoing thrombogenesis as being contributory to the progression and possibly to the initiation of PAD. Aspirin alone did not prevent haemostatic hyperreactivity in these patients and flow-mediated platelet function was the most sensitive and specific indicator of advanced disease. This technique thus appears to be valuable, not only for evaluating therapeutic strategies to prevent platelet activation, but also in elaborating platelet-related mechanisms involved in thrombogenesis and atheroma formation.

Platelets of patients with peripheral arterial disease are hypersensitive to heparin.

We sought to verify earlier reports of increased platelet reactivity in patients with peripheral arterial disease (PAD) during perioperative heparin administration, and to test the hypothesis of platelet hypersensitivity to heparin in these patients. Before and after incubation of platelet rich plasma with unfractionated (UH), low molecular weight heparin (LMWH), and a low molecular weight heparinoid, real-time quantitative assessment of platelet function was performed by stagnation point flow adhesio-aggregometry (SPAA) in 21 patients with PAD and 14 healthy volunteers. With SPAA the occurrence of spontaneous aggregation is pathological. In the 15 patients requiring operation, platelet function and count were measured at regular intervals. To detect heparin dependent antibodies, the heparin induced platelet activation assay (HIPA) was performed preoperatively and after 10 days of heparin therapy. Mean baseline platelet adhesion in patients was double that observed in controls (p < 0.001). Spontaneous aggregation was seen in 9 (43%) patients and no controls (p < 0.001). In controls heparinoid reduced, whereas UH and LMWH slightly increased adhesion. Spontaneous aggregation was observed once with UH. Platelets from patients showed significantly enhanced adhesiveness and aggregability (p < 0.05) with UH and LMWH when compared to controls. Effects with the heparinoid were less pronounced and non-significant. In patients requiring operation, postoperative increases in platelet function and reductions in count were significant (p < 0.001). Ten (67%) experienced a fall in platelet count of > 50%. Preoperatively the HIPA assay showed no evidence of antibodies, whereas after heparin administration antibodies were verified in 4 (32%) patients and could not be ruled out in 6 (40%). Three developed postoperative thrombosis, in one case fatal. A hypersensitive in vitro and in vivo platelet response to heparin was verified in patients with PAD and a large number developed the immunological type of heparin-associated thrombocytopenia. Our findings suggest that a thrombin antagonist which does not interact with platelets may give the best perioperative protection in these patients.

Effect of fibrinogen substitution in afibrinogenemia on hemorheology and platelet function.

Fibrinogen substitution can correct bleeding in afibrinogenemia. We assessed the effect of fibrinogen substitution in a patient lacking immunoreactive fibrinogen. Fibrinogen and thrombin time were not measurable before, but became detectable within 30 min after substitution, parallelled by an increase in ADP-induced platelet aggregation from < 10% to 32%. Platelet adhesion, measured by Stagnation Point Flow Adhesio- Aggregometry, was not detectable prior to substitution but attained normal values thereafter. Scanning electron microscopy of adhering platelets revealed pseudopodia protrusion and spreading. Morphometry revealed two populations of spread platelets one of which demonstrated inhibited spreading as compared to healthy controls. Immunoelectron microscopy revealed normal GPIIb/IIIa receptor expression, both before and after substitution. Dynamic and kinematic viscosity of plasma and whole blood remained below the 99.9% confidence border of a healthy control group. In afibrinogenemia fibrinogen levels as low as 10% of normal concentration sufficed to normalize coagulation, platelet adhesion, and, partially, spreading.

Residence time in niches of stagnant flow determines fibrin clot formation in an arterial branching model--detailed flow analysis and experimental results.

Deposition of blood components in branching flow has been investigated primarily with regard to platelets. We instead examined thrombin-induced fibrin clot formation in separated laminar as well as turbulent branching flow. The most rapid clot growth and largest clot was obtained at the lowest inflow rate. Increased inflow reduced the clot size and turbulence completely prevented clot formation. Examination of corresponding flow conditions revealed the recirculation zone in laminar flow to be characterized by two stationary, counterrotating vortices. Niches of stagnant flow, exhibiting long residence times, low wall shear rates and characterized by convergent flow, were spared between the bulk flow and these vortices. Here, fibrin clot growth continued even when shear rates were increased more than 100-fold. Our results indicate that, in branching flow, the long residence times and convergent flow characteristic of flow niches rather than shear rate are critical for fibrin clot formation.

Platelet response to vascular surgery--a preliminary study on the effect of aspirin and heparin.

Patients with peripheral arterial disease (PAD) demonstrate high cardiovascular mortality, which is further increased after arterial reconstruction. Enhanced platelet reactivity has been postulated for these patients. The effect of surgery and of periprocedural aspirin and heparin therapy on platelet reactivity was assessed with the Stagnation Point Flow Adhesio-Aggregometer (SPAA). The platelet adhesivity and aggregability of 44 PAD patients was quantitated perioperatively. Aspirin was administered during the entire course, low molecular weight heparin (LMWH) preoperatively and as of the fourth postoperative (pOP) day and unfractionated heparin (UH) upon surgery and three days thereafter. A group of 15 aspirin-free general surgical patients receiving LMWH and with no evidence of PAD served as controls. Plasma fibrinogen levels and platelet count were determined. The heparin-induced platelet activation (HIPA) assay for detection of heparin-associated thrombocytopenia (HAT) antibodies was also performed. Baseline values of SPAA-measured platelet reactivity (p < 0.001) and plasma fibrinogen (p < 0.01) were higher for patients as compared to controls and increased markedly after surgery. In the PAD group maximum platelet activation and fibrinogen levels coincided with a marked drop in platelet count and were concomitant to administration of unfractionated heparin. Thereby, a drop in platelet count of > 30% was observed in 25 patients (57%). The HIPA test verified HAT antibodies in 3 (12%) of these patients, two of which suffered postoperative thrombosis. In the control group significant pOP increases were noted only for plasma fibrinogen. Changes in platelet count and reactivity were minimal and nonsignificant. No thrombosis occurred and no HAT antibodies were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow mediated fibrin thrombus formation in an endothelium-lined model of arterial branching.

In vivo arterial thrombosis occurs preferentially at curvatures and branchings, i.e. regions of flow separation and recirculation where blood is retained orders of magnitude longer than within straight vessel sections. To examine the effect of such disturbed flow on endothelial thromboresistance glass T-branchings lined with endothelial cells from human umbilical cord veins (HUVEC) were perfused with buffered fibrinogen solution (3mg/ml). The flow was adjusted to form a large recirculation zone and flow conditions were determined beforehand by means of flow visualization via dye injection as well as by laser ultramicroscope anemometry. Thrombus formation, which was registered on-line by video and evaluated planimetrically, was induced by injection of thrombin at concentrations ranging from 0.3 to 2.0 units/ml. Fibrin thrombus growth always began within the flow niche at the point of flow separation and extended downstream along the wall and into the vessel lumen finally occluding up to 80% of the lumen. Light and electron microscopy revealed that overall thrombus form as well as the orientation of single fibrin fibers were correlated strictly to the prevailing streamlines. Despite the integrity of the endothelial lining fibrin thrombus formation occurred. The fibrin fibers closely contacted the endothelial surface. These results indicate that recirculation zones promote fibrin thrombus formation sufficient to obstruct the vessel lumen and that intact endothelium alone is insufficient in preventing adhesion of fibrin to its surface.

[Increased pre- and postoperative thrombocyte activity in vascular surgery patients]. / Gesteigerte prä- und postoperative Thrombozyten-Aktivität bei gefässchirurgischen Patienten.

To determine the effect of vascular surgery on platelet function, a perioperative investigation of 37 patients with peripheral arterial disease (PAD) was performed using the Stagnation Point Adhesio-Aggregometer (SPAA). The SPAA provides well defined flow conditions. By means of dark field microscopy platelet microthrombus formation can be directly observed and measured continuously. Mathematical evaluation of resulting growth curves renders the constants for adhesion and aggregation, Kpw and Kpp, respectively. The PAD patients were divided into 2 groups: diabetics (n = 9) and nondiabetics (n = 28), and were examined perioperatively at regular intervals (average: n = 8). Preoperatively all patients received aspirin and low molecular weight heparin (LMWH). As of surgery and up to the third postoperative day all patients received unfractionated heparin (UH), at which time LMWH was resumed. Plasma fibrinogen concentration was also determined. Data obtained preoperatively were compared to those of 40 healthy volunteers (without medications). In the present study a significant increase (p < 0.001) in platelet reactivity was verified in PAD patients in spite of aspirin and LMWH administration. As of the first and up to the 8th day after surgery, a marked increase in platelet adhesivity and aggregability as well as plasma fibrinogen concentration and a concomitant decrease in platelet count was observed. Maximum values were obtained during intravenous administration of UH. Thrombocytopenia (< 150,000/ml) was observed in 12 patients. The hypercoagulability response to vascular surgery observed in the present study occurred in spite of therapy with aspirin and heparin. Our findings indicate the need for further improvement in conventional therapy and the SPAA as a useful tool in monitoring the effectiveness of current as well as of future inhibitors of platelet function.

Quantitative analysis of platelet function using stagnation point flow aggregometry. First clinical results.

BACKGROUND: The clinical consequences of atherosclerosis result from vascular occlusion. The central role of platelet-vessel wall interaction in the initiation and perpetuation of this process is well established. Individual analysis and quantification of two major platelet functions underlying atherosclerosis and thrombosis, i.e. adhesion (platelet-wall interaction) and aggregation (platelet-platelet interaction), would contribute significantly towards elucidation of the mechanisms involved and therefore towards optimization of prophylaxis and therapy. The Stagnation-Point-Flow-Adhesio-Aggregometer (SPAA), in which such an evaluation of platelet function is possible, was thus standardized and its clinical reproducibility and predictive power assessed. METHODS: Using the SPAA, a morphometric separation of adhesion and aggregation is obtained via dark field micrographs of platelet microthrombi formed during stagnation point flow of platelet rich plasma (PRP). Quantification is achieved via biomathematical evaluation of simultaneously obtained growth curves, whereby the degree of adhesivity and aggregability is reflected in the respective growth rate constants Kpw (%) and Kpp (%). Experiments with the PRP of 36 healthy volunteers were performed and the results compared to those obtained for 32 patients exhibiting angiographically verified peripheral arterial disease (PAD). RESULTS: The control group exhibited values (Kpw) ranging from 0.40% to 1.10% (average Kpw: 0.71 +/- 0.21%). Differences in average Kpw value between the control subgroup over and that under 45 years of age were absent. A spontaneous platelet aggregation was not observed in the controls (Kpp = 0%). The overall intraindividual Kpw variation in 18 volunteers examined 3 times or more ranged from a minimum of 3% to a maximum of 20% of respective Kpw value. The patients were divided into two subgroups: diabetics and nondiabetics. The nondiabetic group demonstrated an average Kpw of 1.56%. In addition, a spontaneous aggregation was observed in 50% of all experiments (average Kpp = 1.42%). The diabetic group exhibited the highest average adhesion value (Kpw = 1.94%) occurrence of spontaneous aggregation in all experiments (Kpp = 2.10%). CONCLUSION: The consistency in adhesion values obtained among the controls as well as the minimal intraindividual variance observed, demonstrates the reproducibility of the method. The statistically significant increase (p < 0.001) in adhesivity of patients as compared to controls, as well as the common occurrence of spontaneous aggregation can therefore be considered a pathologic platelet response reflecting the severity of the disease. Results obtained verify the presence of circulating hyperreactive platelets in PAD patient and indicate the predictive power of the method. Thus the SPAA may be of considerable aid in improving thrombosis prophylaxis and therapy.