hemostasis in patients with severe von willebrand disease improves after normal platelet transfusion...

T R A N S F U S I O N P R A C T I C E

Hemostasis in patients with severe von Willebrand disease improves after normal platelet transfusion and normalizes

with further correction of the plasma defect

R. Castillo, G. Escolar, J. Monteagudo, J. Aznar-Salatti, J.C. Reverter, and A. Ordinas

BACKGROUND: A defective hemostatic effect of plasma concentrate infusion in patients with severe von Willebrand disease (vWD) has been ascribed to the absence of platelet von Willebrand factor (vWF) STUDY DESIGN AND METHODS: The role of platelet vWF in hemostasis of severe VWD was investigated. A plateletpheresis unit (4-5 x 10” platelets) from a normal compatible donor was transfused before any cryoprecipitate infusion to three type 3 vWD patients and to one patient with severe type 1 vWD with low levels of platelet vWF who required replacement therapy for bleeding episodes. Autologous platelets were transfused to one of the patients with type 3 vWD. RESULTS: Partial corrections of bleeding times (14-17 min vs. baseline >30 min) were observed in all patients after the transfusion of normal platelets. During cryoprecipitate infusion, bleeding times were normalized (c6 min), and bleeding episodes stopped when plasma levels of vWF activity ranged from 14 to 18 U per dL. Plate- let interactions with the subendothelium increased in parallel with the correction of bleeding times. These results indicate that if approximately 20 percent of the total number of platelets have normal vWF antigen and if plasma vWF levels are at least 14 U per dL, then bleeding times will normalize and mucosal hemorrhages will stop. Transfusion of autologous platelets in one patient with type 3 vWD did not modify bleeding times or platelet adhesion on the subendothelium. CONCLUSION: The hemostatic effect of normal platelets in type 3 vWD seems to be related to the platelet vWF in the transfused platelets.

here i s increasing evidence that intraplatelet levels of von Willebrand factor (vWF) correlate bet- ter with bleeding times (BTs) than do plasma vWF T levels. It has been observed that patients with type

1 von Willebrand disease (vWD) who have normal levels of platelet vWF have shorter bleeding times than those with low or dysfunctional platelet vW, despite equally low plasma v W E I - ~ Bone marrow transplantations carried out between pigs with normal and severe vWD have shown that platelet vWF i s necessary for normal primary h e m o s t a ~ i s . ~ ~ ~

m e 3 vWD i s characterized by a lack o f vWF in plasma, platelets, and vessel walls. It has been shown repeatedly that the transfusion of plasma concentrates i s not always SUE- cient to correct BT and to control clinical hemorrhage, although the plasma levels o f vWF and factor VIII activity attained n ~ r m a l i t y . ~ - l ~ The absence o f correction o f the BT in these severely deficient vWD patients occurs even when the vWF multimeric structure i s intact in the plasma concentrate as well as in the corresponding posttransfusion plasma o f the patient.I0-l3 This defective hemostatic effect obtained after the transfusion o f plasma concentrate might be ascribed in part to the absence o f platelet vWF in these severely affected patients. In a recent study, we demonstrated the hemostatic effect o f normal platelet transfusion in patients with severe vWD13 who suffered persistent mucosal hemorrhage and prolonged sk in BTs, although they had attained normal levels o f plasma vWF after cryoprecipitate infusion. The plasma and platelet vWF levels necessary to attain correct hemostasis remain to be determined.

ABBREVIATIONS: BT(s) = bleeding time(s]; PC(s) = platelet concentrate(s); vWD = von Willebrand disease; vWF = von Willebrand factor; vWF:Ag = von Willebrand factor antigen.

From the Servicio de Hemoterapia y Hemostasia. Hospital Clinic, Universidad de Barcelona, Barcelona, Spain.

Supported by grants FIS S92/0695,94/1213, and 95/1080 from the Fondo de Investigaciones Sanitarias de la Seguridad Social.

Received for publication April 17,1996; revision received January 10,1997, and accepted February 12,1997.

TRANSFUSION 1997;37:785-790.

Volume 37, August 1997 TRANSFUSION 785

CASTILLO ET AL.

In the present study, we attempted to determine the hemostatic effect of normal platelet concentrate (PC) transfusion in severe vWD patients. We also investigated the platelet and plasma vWF levels needed to support normal hemostasis in these patients.

MATERIALS AND METHODS Patients The study was performed on three type 3 vWD patients (Pa- tients l, 2, and 3), who had unmeasurable levels ofvWF antigen (vWF:Ag) and vWF activity in plasma (normal values: >60 UldL for both the antigen and the activity), as well as unmeasurable levels of platelet vWFAg (normal range, 0.3- 0.6 UI lo9 platelets). All these patients presented with factor VIII activity of 56 U per dL in plasma (normal range, 60-150 UldL) and a BT longer than 30 minutes. A type 1 vWD patient with low platelet vWF (Patient 4) was included in the study. In this patient, BT was longer than 30 minutes; plasma levels of factor VIII activity, vWFAg, and vWF activity were 8, 6, and c6 U per dL, respectively, and platelet vWFAg was 0.05 U per lo9 platelets.

All the patients were enrolled in our study after a diag- nosis of mucosal hemorrhage was confirmed and replacement therapy was indicated. All patients presented with values of hemoglobin ranging from 11.2 to 13.4 g per dL and hematocrits from 36 to 42 percent. All cases included HLA alloimmunization, surface antigen to hepatitis B, and antibodies to hepatitis c, while human immunodeficiency virus antibodies were detected in two patients. None of the patients had shown inhibitor activity to vWF at the time the study was performed. This group of patients had previously shown on many occasions a poor clinical response to plasma concentrates as assessed by incomplete control of bleeding episodes, despite achieving optimal plasma vWF activities. This study was approved by the Hospital Commit- tee on Human Experimentation. Informed consent was obtained in each case according to the Declaration of Helsinki.

Design of the study Normal PCs were transfused to patients presenting with mucosal hemorrhage that required replacement therapy. PC transfusions were completed in 30 minutes. The platelet doses transfused to each patient were 4 x l o l l (Patient 1),4.5 x 10” (Z), 4.9 x 10” (3), and 5 x 10” (4). Particular care was taken to minimize the volume of plasma infused with the PCs. One hour after the platelet transfusion, standard blood bank cryoprecipitate was injected into the patients by use of an infusion pump at a rate set to provide approximately 20 IU of vWF per kg per hour, assuming an average of 120 IU of vWF per 45 mL in each cryoprecipitate bag. Levels of BT, the multimeric structure of plasma and platelet vWE and the interaction of platelets with vessel wall subendothelium

were determined both before and after platelet transfusion, as well as sequentially every 30 minutes during the cryoprecipitate infusion.

A similar study was carried out in one of the type 3 vWD patients who received autologous platelet transfusion. The presence of vWF associated with the aggregates formed on the subendothelium in the perfusion studies was detected by an indirect immunocytochemical technique using gold particles.

Methods General. PCs were collected by apheresis from a single HLA- matched donor, using a single-pump method (V50-I, Hae- monetics, Braintree, MA). Cryoprecipitate units were pre- pared by the hospital blood bank from single donations of fresh-frozen plasma. The plasma was stored at -80°C and units were pooled in plastic bags before transfusion.

BT was measured with a sterile disposable device (Sim- plate, General Diagnostics, Morris Plains, NJ).16 The procedure and the expression of BT were as previously described.13 The normal range was 3 to 8 minutes.

Measurements of the levels and multimeric structure of vWF were carried out as described,I3 by using an electro- immunoassay method (vWF:Ag), formaldehyde-fixed platelets (vWF activity), and sodium dodecyl sulfate-agarose gel electrophoresis (vWF multimers). Measurements of vWF levels in platelets were performed by assessing vWF levels in washed platelets lysed by 0.1-percent nonionic detergent (Triton X-100, Sigma, St. Louis, MO).

Perfusion studies. Platelet interactions with the vessel wall were studied in the perfusion system developed by Baumgartner and Muggli” and Escolar et al.,I8 by using citrated whole blood and subendothelium of everted segments of rabbit abdominal aorta at a constant shear rate of 1300 per second.

In those studies aiming at assessing improvements of platelet interactions after PCs or cryoprecipitate transfusion, flow was maintained for 10 minutes on vessel segments previously denuded by brief air exposure. More thrombogenic conditions were used in those studies conducted to local- ize vWF in platelet aggregates formed on the subendothelium. Samples of anticoagulated blood were perfused for 30 minutes through annular chambers containing vessel segments in which endothelium had been removed and the collagen in the subendothelium exposed by incubation with a-chymotrypsin.

In all cases, perfused segments were always futed in 4- percent paraformaldehyde in phosphate-buffered saline. Details on the histologic method and morphometric analy- sis procedures have been described p r e v i ~ u s l y . ’ ~ - ~ ~ - ’ ~ In short, platelets interactingwith subendothelium were evalu- ated according to Baumgartner and Muggli,” a computer- ized method was used for the morphometric evaluation,I8 and the deposition of platelets on the subendothelium was

786 TRANSFUSION Volume 37, August 1997

PLATELET vWF AND HEMOSTASIS

expressed as a percentage of the total length of the vessel screened.

Localization of vWF in histologic sections of perfused vessels. The presence of vWF was detected on glycol-meth- acrylate sections of the perfused vessels by an indirect im- mdnocytochemical method. Three pm thick sections were exposed to e primary antibody (rabbit anti-human vWF; Dakopatts AIS, Glostrup, Denmark). The excess of antibody was removed by repeatedly washing the sections with phosphate-buffered saline. The antibody bound to the section was revealed by incubation with protein A gold (10-nm particles, Amersham International, Amersham, UK). Finally, gold particles bound to the sections were made visible in the light microscope by a silver enhancement procedure (In- tense B, lanssen Pharmaceutica, Olen, Belgium). Sections

A >30 30

20 .- E

t- m v

10

were slightly counterstained with toluidine blue and observed by transmitted-light bright-field microscopy.

RESULTS vWF activities and BT After platelet transfusion, BT was partially corrected in the four patients studied. It was shortened to 15 (Patient I), 16 (21, 15 (31, and 14 (4) minutes (baseline >30 min), although plasma vWF activity was not modified (Fig. 1) . During the cryoprecipitate infusion, the BT was normalized (5 [Patient 1],5 [Z], 4 131, and 7 141 min) when approximately 10 IU vWF per kg of body weight was infused. At this point, plasma vWF activity levels attained 18 (Patient l ) , 14 ( Z ) , 15 (3), and 15 (4) U per dL (Fig. l), and mucosal bleeding stopped in all four patients. With further cryoprecipitate infusion, there was a proportional increase in vWF activities (Fig. 1). The plasma levels of vWF:Ag after platelet transfusions and during the cryoprecipitate infusions paralleled those of vWF activity.

As shown in Fig. 2, vWF:Ag levels in platelet lysates obtained from the three type 3 vWD patients after platelet transfusion ranged from 0.07 (Patient l), to 0.05 (Z), and 0.06 (3) U x lo9 platelets (baseline values, c0.015 U x loy). In the type 1 vWD patient with low levels of platelet vWF (Patient 4), the platelet vWF:Ag level after platelet transfusion reached 0.09 U x lo9 platelets (baseline value, 0.05 IJ x lo9 platelets).

Transfusion of autologous platelets to one of the type 3 vWD patients (Patient 1) did not modify either BT or vWF levels. During the subsequent infusion of normal cryoprecipitate, BT remained virtually unmodified (Fig. 1). Plasma

Basal After 10 20 30 40

transfusion During cryo- PC (IU vWF/kg body weight)

precipitate infusion

Fig. 1. BTs (A) and levels of v W F activity (B) in three type 3 vWD patients-Patients 1 (O), 2 (01, and 3 (A)-and in one type 1 vWD patient with low platelet vWF (Patient 4) (0). Val- ues reflect levels before and after PC transfusion and during the following infusion of cryoprecipitate. The series of BTs obtained in one of the type 3 vWD patients (0) after autologous platelet transfusion and during the subsequent cryoprecipitate Infusion are represented by a broken line. Symbols identi- fylng the different patients are used in all figures.

Basal After 10 20 30 40 PC (IU vWF/kg body weight)

transfusion During cryoprecipitate infusion

Fig. 2. Levels of platelet vWAg in the type 3 vWD patients and in a patient with type 1 (low platelet vWF) after normal platelet transfusion and during the following cryoprecipitate Infusion. The type 3 vWD patients are Patient 1,O; Patient 2, 0; and Pa- tient 3, A; the type 1 vWD patient, ..


CASTILLO ET AL.

Basal A R ~ ~ 10 20 30 40

transfusion During cryo- PC (IU vWF/kg body weight)

precipitate infusion

Fig. 3. Changes in platelet adhesion in three type 3 vWD patients and in one type l patient (low platelet vwF) after normal platelet transfusion and during the successive cryoprecipitate infusion. The type 3 vWD patients are Patient 1, 0; Patient 2, 0; and Patient 3, A ; the type 1 vWD patient, Patient 4, 0.

levels of vWF increased in parallel to the amount of cryoprecipitate infused.

Platelet interactions with vessel subendothelium Platelet deposition on the vessel subendothelium, expressed as a percentage of total covered surface, reached values of 10 (Patient 11, 9 (2), 11 (3, and 12 (4) percent after normal platelet transfusion (baseline level, c4%), and platelet aggregates were only occasionally observed.

As shown in Fig. 3, platelet deposition increased up to 11 (Patient l), 14 (Z), 15 (31, and 16 (4) percent (normal values, 21 4%), when infusion of cryoprecipitate had raised plasma vWF activity levels to 18, 15, 14, and 15 U per dL, respectively (Fig. 1). The presence of platelet aggregates was more frequent.

Localization of vWF in the platelet aggregates formed on the subendothelium Positive gold labeling for vWF was observed on platelets lo- cated in the aggregates formed on the more thrombogenic subendothelium (a-chymotrypsin-digested) that was perfused with anticoagulated blood obtained from patients immediately after platelet transfusion (Fig. 4).

DISCUSSION The results obtained in this study show that the transfusion of PCs to three type 3 vWD patients and one type 1 vWD patient with a low level of platelet vWF partially corrected both the BT and the interaction of platelets with subendothelium, although plasma vWF levels remained unchanged. Furthermore, the present studies indicate that plasma lev-

Fig. 4. Localization of vWF in cross-sections of the perfused vessels. vWF was detected with a primary polyclonal antibody and revealed by exposure to protein A gold followed by silver enhancement. The pointed arrow indicates positive labeling of a platelet aggregate formed on a collagen-rich subendothelial surface (IEL = internal elastic lamina). (1200 x).

els of vWF as low as 14 U per dL could be adequate to pro- mote normal hemostasis, provided there was a partial correction of platelet vWE

The correction of plasma vWF defect through plasma concentrate infusion is the therapy of choice for hemorrhagic episodes in vWD patients. However, several studies have reported that the correction of BT and the stopping of mucosal hemorrhage are not complete in type 3 vWD patients, despite normalization of plasma vWF level^^-'^ and the normal multimeric structure of VWF.I~-’~ In a recent study, we observed that normalization of BT could be achieved in such patients when normal platelets were transfused after cryoprecipitate inf~si0n.I~

In our present investigations, we observed that plasma levels of vWF as low as 14 U per dL normalized BT (c6 min) in type 3 vWD patients, provided they had previously received transfusion of normal platelets. Furthermore, studies with blood under flow conditions performed with samples from the same patients showed that adhesion of platelets to the subendothelium increased at the same time as the BT decreased. After normal platelet transfusion, the surface covered by platelets ranged from 9 to 11 percent (baseline level, 4%). When plasma vWF levels reached between 14 and 18 U per dL, the surface covered by platelets ranged from 11 to 16 percent. These results suggest that approximately 20 percent of normal platelets and plasma vWF levels as low as 14 U per dL are sufficient to normalize BT and to stop mucosal hemorrhages.

The partial hemostatic effect obtained after normal platelet transfusion could not be ascribed to a nonspecific activation of the transfused platelets. Flow cytometric studies performed on samples of platelets transfused and on samples of blood taken from the patients after transfusions (data not shown) indicated that transfused platelets did not show signs of activation.

The complete lack of hemostatic effect after transfusion of autologous platelets in one of the type 3 vWD patients


PLATELET vWF AND HEMOSTASIS

indicates that the partial hemostatic effect of normal platelet transfusions to this type of patient is due to the vWF in the platelets transfused. It is important to emphasize that the autologous platelets (4.8 x 10”) were obtained by apheresis with the same system as normal platelets. Thus, the only difference between the two transfused concentrates was the absence or presence of platelet vWF at normal levels.

Localization studies performed on the vessels that had been perfused with blood from the patients after normal platelet transfusion demonstrated the presence of vWF that was related to the platelet aggregates formed on the subendothelium of the perfused vessel wall. This observation supports the possibility that the normal platelets that are transfused become part of the aggregate formed on the subendothelium. Our immunocytochemical studies cannot determine whether the normal transfused platelets inter- acted more efficiently with the subendothelium than the patient’s platelets. However, the incorporation of normal platelets in the aggregates is consistent with the idea that the transfused platelets actively participate in the partial correction of the hemostasis observed in our studies.

Pehsion experiments have greatly contributed to the understanding of the critical role that vWF plays in platelet adhesion.20-21 Several reports have emphasized the impor- tance of subendothelial vWF in supporting platelet adhesion.5s22-25 In the present study, plasma vWF levels (non- endogenously produced) as low as 14 U per dL normalized BT, and controlled hemorrhagic episodes, provided there was a partial correction in platelet vW. These data reinforce the concept that platelet vWF plays an important role during platelet-adhesive e ~ e n t s . ~ ~ , ~ ~ In general, our results are in agreement with the data from animal e~perimentation,~,~ in that platelet vWF would be necessary but not sufficient to warrant normal primary hemostasis.28

The platelet vWF levels attained after normal platelet transfusions in the three patients with severe vWD were similar to those obtained in the baseline platelet sample from the type 1 vWD patient (Fig. 2). These results suggest that the hemostatic effect of platelet vWF in the type 3 vWD patients after the transfusion of normal platelets is more dependent on the presence of some of normal platelets, than on a whole population of platelets with decreased pools of vWE as is the case in the type 1 vWD patient included in our study.

In summary, platelet vWF plays an important role in the hemostatic process by facilitating the accumulation of platelets at the site of vessel wall injury. Information on levels and the function of platelet vWF may be useful for clinical clas- sification of vWD patients according to their hemorrhagic risk and for providing adequate treatment. The transfusion of normal platelets should not be used for routine treatment of vWD, but may be useful in type 3 vWD patients with severe bleeding that is resistant to the standard replacement of plasma vW.

ACKNOWLEDGMENT

The authors thank Esperanza MallafrC for preparation of the manuscript.

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AUTHORS

Ricardo Castillo, MD, Professor of Medicine, Facultad de Medi- cina. Universidad de Barcelona; and Consulting Professor, Servicio de Hemoterapia y Hemostasia, Hospital Clinico y Provin- cial, Villaroel 170, Barcelona 08036, Spain. [Reprint requests]

cina, Universidad de Barcelona; and Head, Section of the Servicio de Hemoterapia y Hemostasia, Hospital Clinico y Provincial.

terapia y Hemostasia, Hospital Clinico y Provincial.

Hemoterapia y Hemostasis, Hospital Clinico y Provincial.

terapia y Hemostasia, Hospital Clinico y Provincial.

cina, Universidad de Barcelona, and Head, Servicio de Hemo- terapia y Hemostasia Hospital Clinico y Provincial.

Gin& Escolar, MD, Associate Professor, Facultad de Medi-

Juan Monteagudo, MD, Staff Member, Servicio de Hemo-

JosC Aznar-Salatti, MD, Research Assistant, Servicio de

Juan Carlos Reverter, MD, Staff Member, Servicio de Hemo-

Antonio Ordinas. MD, Associate Professor, Facultad de Medi-


hemostasis in patients with severe von willebrand disease improves after normal platelet transfusion...

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