Reininger C B, Boeger C A, Steckmeier B, Spannagl M, Scweiberer L
Chirurgische Klinik und Medizinische Klinik des Klinikum Innestadt, LMU München, Germany.
Int Angiol. 1999 Jun;18(2):163-70.
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.
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).
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.
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.
在周围动脉疾病（PAD）中，动脉粥样硬化呈弥漫性且血栓形成风险很高。我们不仅已表明PAD患者的血小板具有高反应性且对阿司匹林耐药，而且最近还证实它们对肝素也高度敏感。在本研究中，我们已开始阐明这些经常观察到的临床发现背后的机制。
采用传统的ADP激发聚集试验和停滞点血流黏附-聚集测定法（SPAA）检测血小板功能。SPAA可在生物学相关的血流条件下对血小板黏附和聚集进行实时、定量评估。对一名先天性无纤维蛋白原血症女性患者静脉注射纤维蛋白原替代前后的血小板进行了分析。在14例PAD患者和14名对照者中，在与两种血小板膜糖蛋白（GP）-IIb/IIIa抑制剂Ro 43-8857和7E3孵育前后评估血小板反应性。最后，在向富含血小板血浆和来自4名健康志愿者的凝胶过滤血小板（GFP）溶液中添加源自4例PAD患者的血浆等分试样前后进行了实验。在纤维蛋白原替代前，无纤维蛋白原血症患者的血小板在SPAA系统中无法黏附，且最大ADP激发聚集率低于10%。替代后，测得正常的血小板黏附，且激发聚集增加了两倍。患者群体的平均基线黏附是对照组的两倍（p<0.001）。在10例患者中观察到SPAA测定的自发聚集，而对照组均未观察到（p<0.001）。
在最低底物浓度（0.1微摩尔Ro 43-8857，1微克/毫升7E3）时，SPAA测定的聚集和激发聚集均被消除。在这些浓度下，患者的黏附分别降低了65%和40%，对照组分别降低了55%和25%。在0.5微摩尔Ro 43-8857和10微克/毫升7E3时，两组的黏附均完全消除。患者和对照组对抑制剂的血小板反应相似，聚集和黏附之间的剂量反应差异也相似。向志愿者富含血小板血浆中添加患者血浆后，所有4名健康个体的血小板均自发聚集，且该组的平均黏附性增加了两倍。当重新添加到自身血浆中时，GFP的总体黏附能力降低，而在存在患者血浆的情况下，黏附显著增加。
基于这些发现，我们得出以下结论：1）SPAA可测量和量化血小板与液相（聚集）和固定化（黏附）纤维蛋白原的相互作用；2）这些反应由GP IIb/IIIa受体复合物介导；3）血小板黏附的结合亲和力、代谢途径和信号转导不同于聚集所涉及的（可能反映了它们在止血中的不同作用）；4）PAD患者功能正常的血小板在体内被循环血浆成分激活，这导致活化的GP IIb/IIIa在血小板表面的募集增加，从而使反应性总体增加。
