I.血小板、血小板微小顆粒與白血球之交互作用之研究;II.輸血病人血小板抗體之研究

Abstract

血小板與白血球間的交互作用對於血栓形成過程或發炎反應都具有重要的地位。第一部份的實驗即著重於研究血小板與其形成之血小板微小顆粒(platelet microparticle)與白血球間的交互作用。我們利用全血實驗的模式，探討各種血小板醣蛋白(glycoprotein)IIb/IIIa抑制劑對於血小板和白血球交互作用時所形成之血小板和白血球聚集(platelet-leukcoyte aggregate)，白血球的活化，與白血球細胞表面組織因子(tissue factor)表現量等的影響。在以ADP刺激下的全血實驗，血小板醣蛋白IIb/IIIa抑制劑會加強血小板與嗜中性白血球(neutrophil)聚集形成的量與百分比；也會提昇嗜中性白血球表面表現之CD11b的量。相對地，醣蛋白IIb/IIIa抑制劑對於血小板與單核白血球聚集量則是出現抑制現象。然而，在以ADP刺激之富含血小板血漿(platelet-rich plasma)，醣蛋白IIb/IIIa抑制劑反常地(paradoxically)增加血小板表面P-selectin之表現量。使用對P-selectin與PSGL-1具抑制作用的單株抗體，均可以有效的抑制全血以ADP刺激時白血球CD11b表現增多的情形與血小板白血球聚集增多之情形。使用抑制GPIb-Mac-1交互作用之蛇毒akistin則可以抑制血小板和白血球聚集形成但是對於白血球CD11b表現增加之現象並無明顯抑制作用。 血小板在受到活化時，其細胞表面會有微小粒狀物脫落形成所謂血小板微小顆粒。這些血小板微小顆粒表面也帶有未活化前血小板表面各種特有抗原；在研究血小板與白血球交互作用之同時，也值得研究血小板微小顆粒與白血球交互作用是否有所異同。此外，以往之報告極少提及GPIb-Mac-1在於血小板微小顆粒與白血球交互作用之角色，因此也值得吾人進一步探討。在程度近似於靜脈的shear stress下，血小板微小顆粒可以作為嗜中性白血球聚集(neutrophil aggregate)形成的媒介，顯示血小板微小顆粒可以促進嗜中性白血球聚集的形成，此一現象在某些疾病中具有臨床意義。血小板醣蛋白Ib抑制性單株抗體如AP1及SZ2可抑制上述之嗜中性白血球之聚集現象，同時也對血小板微小顆粒所引起的白血球活化作用，β2 integrin表現增加，吸附後superoxide之產生，與對固相fibrinogen之附著作用等均有抑制的效果。這些結果顯示，在血小板微小顆粒與白血球間之作用，GPIb-Mac-1具有一定參與的角色。 在研究血小板微小顆粒參與組織因子移轉(transfer)的實驗中，吾人繼續以GPIb-Mac-1為研究對象，探討其在血小板微小顆粒移轉組織因子至單核白血球中的角色。以collagen刺激過之platelet-rich plasma與之前離心時沈澱下之紅血球與白血球細胞製備之實驗中，表現之組織因子與血小板特有之CD42a之單核白血球數目有增加之情形。這一現象可以用P-selectin之單核抗體9E1或是血小板GPIb抑制抗體AP1, SZ2所抑制。在分離細胞之實驗，血小板微小顆粒具有移轉組織因子至單核白血球或類單核細胞(monocytoid cells)，這些增加的組織因子的量與其procoagulant活性具有正相關，顯示所移轉之組織因子是具有活性。使用抑制P-selectin-PSGL-1或是GPIb-Mac-1之交互作用之單株抗體則可以抑制其組織因子表現量與其procoagulant之活性。 本研究之另一重點則是在於研究多次輸血病人體內形成血小板抗體之情形。經常輸注血小板可以引發所謂異體致敏現象(alloimmunization)，其形成之血小板抗體則可以分為對抗人類白血球抗原(human leukocyte antigen, HLA)與血小板特異抗體，後者其抗體特異性之標的即是血小板上之各種醣蛋白。首先針對103位多次輸注血小板之患者進行研究，利用ELISA的方法，我們發現其中22位病患體內同時有HLA抗體與血小板特異抗體，有12位有HLA抗體，而有5位體內只有血小板特異抗體。血小板特異抗體的檢測通常發生於體內己有HLA抗體之病患為多，而血小板特異抗體之特原標的則主要是對抗血小板GPIa/IIa與IIb/IIIa為主。 在另一實驗中，則是針對長期輸注紅血球之海洋性貧血(thalassemia)病患探討其發生血小板抗體致敏現象之情形。以往針對類似長期以輸注紅血球為主之病患，很少人研究其體內血小板抗體致敏之情形，相關之資料也厥如。在60位海洋性貧血的患者，第一年的檢測結果發現，有19位病患有HLA抗體，13位病患同時有HLA與血小板特異抗體，一位有血小板特異抗體，在一年後的追蹤後發現，有7位病形患在追蹤期間形成了有HLA抗體，HLA抗體消失的有一位；有12位病患其血小板特異抗體曾消失。本實驗証實了紅血球輸血也會有血小板抗體致敏現象，病患在長期輸血過程中形成HLA抗體與血小板特異抗體，而血品中殘留之白血球與血小板則是致敏原的可能來源。

Platelet-leukocyte interaction plays a crucial role in various thrombosis and inflammation processes. The first part of our study focused on the interaction of platelets and their microparticles (MPs) with leukocytes. In a whole blood model, we investigated the effects of various glycoprotein (GP) IIb/IIIa inhibitors on the formation of platelet-leukocyte aggregate, leukocyte activation and tissue factor expression on leukocytes. GPIIb/IIIa inhibitors enhanced platelet-neutrophil aggregate, both in percentage and adhered platelet mass, and the neutrophil CD11b expression in ADP-stimulated whole blood. In contrast, GPIIb/IIIa inhibitors reduced the mass of platelets attached to monocytes in ADP-stimulated whole blood. However, GPIIb/IIIa inhibitors enhanced paradoxically platelet P-selectin and tissue factor expression in ADP-stimulated platelet-rich plasma. The ADP-induced CD11b expression and platelet-leukocyte aggregate were effectively blocked by monoclonal antibodies against P-selectin and PSGL-1. Blockade with the GPIb-Mac-1 interaction with agkistin, a snake venom of GPIb antagonist, inhibited platelet-leukocyte aggregate but inhibited less effectively the CD11b expression of leukocytes. Since platelet MPs are membrane vesicles shed by platelets after activation, and carry antigens characteristics of intact platelet, it is interesting to delineate the similarities between platelet MP-leukocyte interaction and platelet leukocyte interaction. Besides, few studies addressed role of the GPIb-Mac-1 in mediating platelet MP and leukocyte adhesion. In our study we found that platelet MPs can serve as a bridge in supporting neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in disease where the platelet MPs are elevated. Platelet GPIb monoclonal antibodies, AP1 and SZ2, can block the aggregation. These antibodies also decreased platelet MP mediated-neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP mediated neutrophil adherence to immobilized fibrinogen. These data provide the evidence that the involvement of GPIb-Mac-1 in the cross-talk between platelet MPs and neutrophils. In another study, we further explode the role of GPIb-Mac-1 binding in mediating tissue transfer from platelet MP to monocyte. Incubation of plasma obtained from collagen-stimulated platelet-rich plasma with a sediment of red and white blood cells resulted in an increase in the number of monocytes that express tissue factor (TF) and platelet specific antigen CD42a. This increase can be effectively reduced in the presence of P-selectin blocking monoclonal antibodies 9E1, and also GPIb blocking antibodies AP1 and SZ2. In isolated cell experiments, platelet MPs also transferred TF to monocytes or THP-1 monocytoid cells. The increased surface TF expression was highly correlated with the procoagulant activity. Blocking the P-selectin-PSGL-1 or GPIb-Mac-1 cross talk with inhibitory monoclonal antibodies significantly inhibited the increased TF and the associated procoagulant activity. The other part of our studies focused on the study of platelet antibody on mutilply transfused patients. Frequent platelet transfusions may induce alloimmunization status. Platelet alloimmunization involves the formation of human leukocyte antigen (HLA) antibodies and platelet-specific antibodies, the latter being aimed at platelet GPs as antigens. In the first serial study, one hundred and three patients who had received multiple platelet transfusions were enrolled and were tested for the presence of platelet antibodies. ELISA study showed that 22 patients had both HLA and platelet-specific antibodies, 12 patients had HLA antibodies alone, and five patients produced platelet-specific antibodies in the absence of HLA antibodies. Most of the platelet –specific antibodies were found among patients who had HLA antibodies. The most frequently involved platelet GP antigens were Ia/IIa and IIb/IIIa. In the second serial study, we investigated platelet alloimmunization status after long term red cell transfusion in thalassemia patients. Few data have been available to address the platelet alloimmunization on a group of patients who received long-term red cell transfusion. Sixty thalassemia patients were included in this study. At first year of the study, 19 patients had HLA antibodies, 13 had HLA antibodies and platelet specific antibodies, and one had platelet specific antibodies. The follow-up study showed that 7 patients developed HLA antibodies, while one patient lost HLA antibody activity; 9 patients developed new platelet-specific antibodies, and 12 patients lost at least one of their platelet-specific antibodies. This study confirmed that lone term red cell transfusions can induce platelet alloimmunization, both to HLA antigens and platelet specific antigens. The residual platelets and white blood cells in red cell component could be the sources of immunization. We also found that, HLA antibodies likely sustain longer than platelet specific antibodies.