A Prospective Study of G-CSF Effects on Hemostasis in Allogeneic Blood Stem Cell Donors

Bone Marrow Transplantation, (1999) 23, 991–996  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt A prospective study of G-CSF effects on hemostasis in allogeneic blood stem cell donors

R LeBlanc1, J Roy1, C Demers1,LVu2 and G Cantin1

1St Sacrement Hospital, Laval University, Quebec City; and 2St Sacrement Hospital, Quebec City, Quebec, Canada

Summary: The last few years have witnessed an important increase in the use of peripheral blood stem cells (PBSC) for allogeneic Granulocyte colony-stimulating factor (G-CSF) is used in transplantation. Granulocyte colony-stimulating factor (G- healthy donors of peripheral blood stem cells (PBSC) for CSF) is currently the most widely used cytokine for stem allogeneic transplantation. However, some data have cell mobilization. Exposing a large number of otherwise recently suggested that G-CSF may induce a hypercoag- healthy individuals to G-CSF raises concerns about poten- ulable state, prompting us to study prospectively 22 tial adverse effects.1 Although usually well tolerated, G- PBSC donors before and after G-CSF 5 ␮g/kg twice CSF administration commonly induces side-effects such as daily. We sought evidence for changes in the following bone pain, headaches, fatigue and nausea. Anxiety, noncar- parameters: platelet count, von Willebrand factor anti- diac chest pain, myalgia, insomnia, fever, night sweats, skin gen (vWF:Ag) and activity (vWF activity), ␤-thrombo- rash, anorexia, dizziness, weight gain, local reactions at the globulin (␤-TG), platelet factor 4 (PF-4), platelet acti- injection site and vomiting have been infrequently reported. vation markers (GMP-140 and PAC-1), activated partial G-CSF has been extensively used for the treatment of neu- thromboplastin time (aPTT), prothrombin time (PT), tropenia following chemotherapy, but short- and long-term coagulant factor VIII (FVIII:C), thrombin–antithrombin side-effects of G-CSF in normal individuals are not well complex (TAT), prothrombin fragment 1+2(F1+2), defined. thrombomodulin (TM) and tissue plasminogen activator In a few small studies, G-CSF was found to induce plate- antigen (tPA:Ag) prior to G-CSF and immediately before let activation. Shimoda and others have shown that G-CSF leukapheresis. ADP-induced platelet aggregation studies receptors are present on the surface of platelets.2,3 These were also performed. G-CSF administration produced receptors appear to be functional in vitro2 and in vivo4,5 in only mild discomfort. We found a significant increase in healthy volunteers. However, the consequences on hemo- vWF:Ag (from 0.99 ؎ 0.32 U/ml to 1.83 ؎ 0.69 U/ml; P stasis of these findings are conflicting. Other investigators Ͻ 0.001), in vWF activity (from 1.04 ؎ 0.34 U/ml to have also observed an increase in platelet aggregation, in U/ml; P Ͻ 0.001) and in FVIII:C (from platelet activation, in von Willebrand factor and in 0.50 ؎ 1.78 U/ml to 1.73 ؎ 0.57 U/ml; P Ͻ 0.001) after thrombin–antithrombin complex after G-CSF, but these 0.37 ؎ 1.12 G-CSF. Of note, four donors with low baseline vWF had results could not be confirmed.2,4–10 a two- to three-fold increase after receiving G-CSF. G- To date, two cases of thrombosis occurring in normal CSF had no impact on the platelet count, ␤-TG, PF-4, donors have been reported.11 The first one was a healthy GMP-140 or PAC-1. The final% of platelet aggregation 54-year-old woman who developed a cerebrovascular acci- decreased from 73 ؎ 22% to 37 ؎ 26% after G-CSF (P dent 2 days after completing an uneventful stem cell Ͻ 0.001). We found a significant decrease in aPTT after donation. The second one was a 64-year-old man with a -G-CSF (29.9 ؎ 3.1 s to 28.3 ؎ 3.3 s; P = 0.004), but the history of coronary artery disease who experienced a myo PT was unaffected. In addition, we also observed a sig- cardial infarction shortly after PBSC collection. nificant increase in TAT, F1+2 and TM, but not in These clinical and biological observations raise concerns tPA:Ag. Our data suggest that G-CSF may possibly about the possibility that G-CSF might in fact induce a hy- induce a hypercoagulable state by increasing levels of percoagulable state in healthy donors and consequently, FVIII:C and thrombin generation. In contrast to this increase the risk of thrombosis. In order to better define the information, we found reduced platelet aggregation after effects of G-CSF on hemostasis, we undertook a prospec- G-CSF administration. The clinical implications of these tive study in healthy PBSC donors focusing on short-term findings remain unclear and larger studies are defi- adverse effects and on hemostatic changes immediately nitely required. after G-CSF administration. Keywords: granulocyte colony-stimulating factor; plate- let activation; thrombosis; healthy peripheral blood stem cell donors Materials and methods

Study design Correspondence: Dr R LeBlanc, CHA, Pavillon Saint-Sacrement, 1050, chemin Sainte-Foy, Que´bec (Qc), Canada G1S 4L8 This is a cohort study, conducted at St Sacrement Hospital, Received 27 July 1998; accepted 22 December 1998 Laval University, Quebec City, Canada. It was approved Effect of G-CSF on hemostasis in allogeneic blood stem cell donors R LeBlanc et al 992 by the institutional review board and written consent was coagulant factor VIII (FVIII:C), von Willebrand factor anti- obtained from all healthy donors prior to participation. gen (vWF:Ag) and activity (vWF activity), thrombin– antithrombin complex (TAT), prothrombin fragment 1+2 + Patients (F1 2), thrombomodulin (TM) and tissue plasminogen acti- vator antigen (tPA:Ag) assays were performed on plasma Between July 1996 and April 1998, all consecutive healthy obtained from anticoagulated blood with citrate (Becton allogeneic PBSC donors were considered for enrollment in Dickinson, 0.105 mol/l) and centrifuged at 2500 g for 15 the study. All donors studied were used as their own con- min at 4°C. Plasma samples were analyzed immediately or trol. Donors were excluded if they had an abnormal platelet stored in aliquots at Ϫ80°C until tested. For assaying, count, bleeding time, activated partial thromboplastin time samples were thawed for 15 min in a 37°C water-bath. The (aPTT) or prothrombin time (PT) prior to G-CSF. Other Monovette blood collection system (Sarstedt, NC, USA) exclusion criteria were a history of unstable angina, myo- containing sodium citrate (0.105 mol/l) was used for plate- cardial infarction, ischemic or thrombotic stroke in the past let aggregation. Platelet-rich plasma was obtained by centri- 3 months, active neoplasia, active inflammatory disease, fugation at 250 g for 6 min at 27°C and was adjusted with pregnancy or a contra-indication to blood donation. platelet-poor plasma (2500 g for 15 min at 27°C) to a plate- let count of 200 × 109/l. Intervention Assays: Ivy bleeding time was performed with the Simp- All donors were evaluated initially by a physician and a late IIR device (Organon Teknika, Durham, NC, USA) with nurse with a complete medical history and physical examin- longitudinal incisions. Platelet counts were measured on a ation. At their first visit, they were instructed on how to Coulter STKS (Miami, FL, USA). Platelet activation mark- administer their medication and on the potential side- ers (GMP-140 and PAC-1) were measured by flow cytome- effects. Before the first dose of G-CSF, blood samples were try with argon laser generating laser light at 488 nm drawn for laboratory evaluation. All donors received G- (Coulter Epics XL-MCL) using 20 ␮l of anti-P-selectin (Pe, CSF (Neupogen, Amgen, Mississauga, Canada) at 5 ␮g/kg B-D) fluorescing at 575 nm and 20 ␮l anti-gpIIb-IIIa subcutaneously twice daily for nine consecutive doses. (FITC, B-D) fluorescing at 525 nm, respectively. Platelet After the ninth dose on day 5, blood samples were drawn aggregation was performed on the PACKS-4 platelet aggre- for the same assays immediately before undergoing leu- gometer (Helena Laboratories, Beaumont, TX, USA) using kapheresis. A personal diary was provided to all donors in 3 ␮mol/l ADP (Boehringer Mannheim, Indianapolis, IN, order to record prospectively any side-effects experienced USA). PT and aPTT were carried out on the Behring during the period of G-CSF administration. Side-effects Fibrintimer A (BFA, Behringwerke, Marburg, Germany) were graded by one investigator after discussion with the using Thromborel-S (Behring) and Automated APTT donor on a scale of 1 to 4; grade 1 were mild side-effects (Organon Teknika) respectively. FVIII:C was measured by with no intervention needed; grade 2 were moderate side- one stage clotting assay on the BFA instrument using effects needing some intervention (acetaminophen to immunoadsorbed FVIII deficient plasma (Dade, Miami, FL, relieve bone pain or fever) but not interfering with drug USA). vWF:Ag, ␤-TG, PF-4, TM and tPA:Ag were perfor- administration; grade 3 were severe side-effects interfering med by enzyme-linked immunosorbent assays (ELISA) with normal daily activity or with the need to discontinue using Asserachrom kits (Diagnostica Stago, Asnie`res-sur- medication; grade 4 were life-threatening complications Seine, France). vWF activity was determined using IMUB- related to the medication. In order to attribute symptoms IND vWF ELISA kit (American Diagnostica, Greenwich, only to G-CSF administration, all donors were instructed CT, USA). Enzygnost ELISA kits (Behringwerke) were to start recording symptoms in their diary 5 days prior to used for TAT and F1+2 determinations. ELISA assays were G-CSF administration. all carried out on CODA Automated EIA Analyzer (Bio- Rad, Hercules, CA, USA). Normal pooled plasma (NPP) Laboratory testing was used for calibration with a defined value of 1 U/ml. Normal values for each test were established on 50 normal Blood collection: Thirty-five to 40 ml of blood were col- subjects and were defined as the mean Ϯ 2 standard devi- lected from each donor before G-CSF and after 96 h, ations (s.d.). The following tests were not performed in the immediately before leukapheresis. EDTA anticoagulated donors who were taking antiplatelet agents (acetylsalicylic blood was used for platelet count and to look for specific acid, ticlopidine, nonsteroidal anti-inflammatory drugs) at markers expressed on activated platelets: (1) anti-P-selectin the time of enrollment: platelet aggregation tests and the against alpha-granule membrane glycoprotein (GMP-140) assays for ␤-TG, PF-4, GMP-140 and PAC-1. and (2) anti-glycoprotein (gp) against the activated con- figuration of gp IIb–IIIa complex (PAC-1). Cooled Diatube Statistical analysis H CTAD containing a mixture of citrate, theophylline, adenosine and dipyridamole (Becton Dickinson, Meylan, Mean levels Ϯ 1 s.d. were calculated for platelet count, France) was used to collect blood for ␤-thromboglobulin vWF:Ag, vWF activity, ␤-TG, PF-4, GMP-140, PAC-1, (␤-TG) and platelet factor 4 (PF-4) assays according to the PT, aPTT, VIIIc, percentage of final and maximal platelet Diagnostica Stago protocol: after 30 min in an ice/water aggregation, TAT, F1+2, TM, tPA:Ag before and after G- bath, blood was centrifuged at 13 000 g for 30 min at 4°C CSF administration. Those values were compared using the and the plasma was stored at Ϫ80°C until used. aPTT, PT, Student’s paired t-test. Effect of G-CSF on hemostasis in allogeneic blood stem cell donors R LeBlanc et al 993 Results results obtained from these four donors, the difference for vWF:Ag and vWF activity, prior and after G-CSF, remains Patients significant. Of importance, vWF:Ag and vWF activity were higher in all donors after G-CSF. Twenty-four consecutive healthy individuals were evalu- The aPTT was significantly shortened from 29.9 Ϯ 3.1 s ated during the study period. Two donors were excluded prior to 28.3 Ϯ 3.3 s after G-CSF (P = 0.004). We observed (one because of inadequate blood samples and the other a significant rise in the levels of TAT, F1+2 and TM after had active rheumatoid arthritis), leaving 22 donors for G-CSF with mean levels before and after of 2.30 Ϯ 0.57 evaluation. There were 10 females and 12 males, aged from to 4.48 Ϯ 4.28 ␮g/l (P = 0.022), 0.76 Ϯ 0.24 to 20 to 63 years (mean 40). Of these 22 donors, two were 1.52 Ϯ 0.61 nmol/l (P Ͻ 0.001) and 26.7 Ϯ 11.3 to on ASA at the initial visit and were subsequently excluded 60.8 Ϯ 17.9 ng/ml (P Ͻ 0.001), respectively. We found no from the analysis of the platelet functional assays. effects of G-CSF on the PT and the tPA:Ag levels (Table 2). Adverse effects In order to document the effects of G-CSF on platelet function, we studied the platelet count, ␤-TG, PF-4 and two Side-effects experienced by donors while on G-CSF are surface markers of platelet activation: GM-140 and PAC- shown in Table 1. G-CSF was well tolerated by all donors. 1. None of these variables were significantly different after Graded side-effects were low in intensity (grade 2/4 or G-CSF. In contrast, we observed a significant decrease in less): bone pain was present in 82% of the donors, mainly the final percentage of ADP-induced platelet aggregation in the pelvis, and totally or partially relieved by acetamin- after 8 min using ADP at a concentration of 3 ␮m: aggre- ophen; fatigue and malaise in 64%; headache in 55%; myal- gation decreased from 73 Ϯ 22% to 37 Ϯ 26% following gia in 41%. Less frequently reported symptoms included G-CSF (P Ͻ 0.001) (Table 2 and Figure 1). Similar results fever (27%), nausea (18%), cough (14%) and dyspnea were observed using the maximal % of platelet aggregation (9%). However, some complaints were also present in (decrease from 78 Ϯ 17% to 50 Ϯ 16%; P Ͻ 0.001). donors before G-CSF administration, at the same intensity (dyspnea in 9%, fatigue and malaise, myalgia and cough in 5%) (Table 1). No donor had to discontinue G-CSF Discussion administration due to side-effects. No bleeding or throm- botic events were reported during or after G-CSF. G-CSF is widely used for PBSC mobilization in allogeneic transplantation. Increasing numbers of otherwise asympto- Effect of G-CSF on hemostasis matic donors are being exposed to a short course of G- CSF. Recent findings of G-CSF receptors on the surface of All donors had a normal platelet count, as well as bleeding platelets2,3 and a few reported cases of acute arterial throm- time, PT and aPTT prior to G-CSF. As shown in Table 2 bosis in patients receiving G-CSF support the hypothesis and Figure 1, a significant rise in the level of vWF:Ag, that a transient hypercoagulable state may be induced by vWF activity and FVIII:C was observed: vWF:Ag G-CSF. In 1992, Conti et al12 reported the first case of increased from 0.99 Ϯ 0.32 U/ml to 1.83 Ϯ 0.69 U/ml (P Ͻ Ϯ Ϯ acute thrombosis in the left popliteal artery of a 69-year- 0.001), vWF activity from 1.04 0.34 U/ml to 1.78 0.50 old man without risk of cardiovascular disease, receiving U/ml (P Ͻ 0.001), and FVIII:C from 1.12 Ϯ 0.37 U/ml to Ϯ Ͻ G-CSF following chemotherapy for bladder carcinoma. The 1.73 0.57 U/ml (P 0.001). Four donors presented low authors assumed that hyperleukocytosis (leukocyte count of baseline values (Ͻ 0.7 U/ml) of vWF:Ag and vWF activity 70.5 × 109/l) induced by G-CSF, with a presumably sub- prior to G-CSF, but none had a bleeding history. G-CSF sequent hypercoagulable state, was a contributing factor for induced a two- to three-fold increase in all four of them: the occurrence of thrombosis. In 1996, Kawachi et al13 vWF:Ag increased from 0.49 Ϯ 0.11 to 1.11 Ϯ 0.31 U/ml reported the case of a 44-year-old man with a non-Hodg- (P = 0.012) and vWF activity from 0.48 Ϯ 0.11 to Ϯ = kin’s lymphoma, who presented with an acute thrombosis 1.08 0.29 U/ml (P 0.011). Even after exclusion of the in the distal aorta. He was receiving G-CSF for chemo- therapy-induced neutropenia and had no predisposing car- diovascular factors. After the patient was re-challenged Table 1 Side-effects experienced by healthy donors before and during with rhG-CSF, an increase in ADP- and collagen-induced G-CSF administration. platelet aggregation was demonstrated. The authors postu- lated that both a rapid increase in the patient’s platelet count Side-effects Before G-CSF During G-CSF n = 22 (%) n = 22 (%) and a direct aggregating effect of G-CSF were responsible for thrombosis. In addition, other cases of arterial throm- Bone pain 0 18 (82) bosis or other vascular complications have been reported Fatigue and malaise 1 (5) 14 (64) in patients who were also receiving chemotherapy concur- Headache 0 12 (55) rently with G-CSF.14 To date, only two case reports of Myalgia 1 (5) 9 (41) acute arterial thrombosis occurring in healthy donors that Febrile state 0 6 (27) 11 Nausea 0 4 (18) might be related to G-CSF have been published. Cough 1 (5) 3 (14) In this study, G-CSF was well tolerated with no signifi- Dyspnea 2 (9) 2 (9) cant adverse reaction. We found that G-CSF at 5 ␮g/kg twice daily for nine doses induces a significant increase in Effect of G-CSF on hemostasis in allogeneic blood stem cell donors R LeBlanc et al 994 Table 2 Hemostatic parameters before and after G-CSF administration in healthy donors

Parameters Before G-CSF After G-CSF P valuea mean Ϯ s.d. mean Ϯ s.d.

Platelet count (×109/l) 232 Ϯ 47 236 Ϯ 73 0.46 vWF:Ag (U/ml) 0.99 Ϯ 0.32 1.83 Ϯ 0.69 Ͻ0.001 vWF activity (U/ml) 1.04 Ϯ 0.34 1.78 Ϯ 0.50 Ͻ0.001 ␤-thromboglobulin (IU/ml) 47 Ϯ 40 45 Ϯ 31 0.92 Platelet factor 4 (IU/ml) 14 Ϯ 29 13 Ϯ 21 0.84 GMP-140 (% of positive fluorescence) 0.2 Ϯ 0.6 0.4 Ϯ 0.6 0.07 PAC-1 (% of positive fluorescence) 0.7 Ϯ 1.1 1.0 Ϯ 1.2 0.57 Platelet aggregation with ADP3 ␮m at 8 min (%) 73 Ϯ 22 37 Ϯ 26 Ͻ0.001 maximal (%) 78 Ϯ 17 50 Ϯ 16 Ͻ0.001 INR 1.01 Ϯ 0.07 1.01 Ϯ 0.09 1.0 aPTT (s) 29.9 Ϯ 3.1 28.3 Ϯ 3.3 0.004 FVIII:C (U/ml) 1.12 Ϯ 0.37 1.73 Ϯ 0.57 Ͻ0.001 TAT (ng/ml) 2.30 Ϯ 0.57 4.48 Ϯ 4.28 0.02 PF1+2 (nmol/l) 0.76 Ϯ 0.24 1.52 Ϯ 0.61 Ͻ0.001 Thrombomodulin (ng/ml) 26.7 Ϯ 11.3 60.8 Ϯ 17.9 Ͻ0.001 tPA:Ag (ng/ml) 0.62 Ϯ 0.30 0.67 Ϯ 0.14 0.53

aCalculated using Student’s paired t-test.

vWF:Ag which correlates with vWF activity. vWF is both III and protein C have higher levels of F1+2 than normal secreted by the platelet ␣-granules and the endothelial individuals.19,20 In addition, oral anticoagulants suppress Weibel–Palade bodies. Our findings do not support a rise thrombin generation with a significant reduction in in vWF derived from platelet secretion, because it is not F1+2.21,22 Although it has been suggested that among associated with a concomitant increase in plasma ␤-TG, patients with a hypercoagulable state, higher levels of F1+2 PF-4, GMP-140 and PAC-1. In contrast, the rise in TM, could identify a subgroup of patients with higher risk of which is secreted by endothelial cells, may suggest an endo- thrombosis, this observation remains to be proven. There- thelial origin for vWF. However, we have not observed a fore, although we documented an increase in F1+2 and concomitant rise in tPA:Ag which is also derived from TAT levels in patients receiving G-CSF, the clinical sig- endothelial cells. It is possible that because of its very short nificance of this finding remains unclear. half-life, the plasma level of tPA:Ag is too temporary to Our finding of a decrease in platelet aggregation after G- be representative of endothelial secretion. At the present CSF is surprising considering that previous studies have time, the exact origin and mechanism responsible for the reported either no significant change or an increase in plate- increase in vWF remains unexplained. let aggregation, and that the clinical events reported were It is possible that the rise in vWF may be unrelated to arterial thrombosis. However, the timing of testing might G-CSF administration, but result from psychological or be important since it has been demonstrated that platelet emotional stresses associated with the procedure. Hemo- aggregation increases when tested soon after G-CSF (within static changes have been documented following acute men- 18 h),3–6 while no change is seen when it is tested at 48 tal stress with a significant rise in vWF level.15 PBSC and 96 h.7 In our study, platelet aggregation was tested donation to a family member is clearly a stressful situation slightly more than 96 h after G-CSF, and we demonstrate for many individuals but, in our opinion, the constant and a decrease in platelet aggregation that was very consistent significant rise in the level of vWF observed in our study in all patients but one. A phenomenon of ‘platelet exhaus- argues in favor of a direct role for G-CSF. However, no tion’ was reported after strenuous exercise of long dur- matter how the increase in the level of vWF observed in ation,23 during hemolytic-uremic syndrome,24 and during our study is produced, a review of patients receiving pre-eclampsia.25 Whether this occurs in our patients also desmopressin as a hemostatic drug during surgery demon- remains unclear. strate that it does not increase the incidence of thrombosis In summary, we found that normal individuals receiving despite a significant rise in the level of vWF associated G-CSF to mobilize hematopoietic stem cells have an with its administration.16,17 Therefore, the rise in vWF, increase in vWF, FVIII:C and evidence of thrombin gener- by itself, is not sufficient to explain a predisposition to ation. Although this may indicate that G-CSF induces an thrombosis. hypercoagulable state, the correlation with clinical throm- In our study, we also demonstrate a statistically signifi- bosis has yet to be proven. Large numbers of healthy cant rise in FVIII:C, TAT and F1+2 after G-CSF. In one donors exposed to G-CSF will need to be evaluated in order study, FVIII:C levels exceeding 1.5 U/ml were associated to determine the risk of thrombosis in this population, and with a six-fold increase in the risk of thrombotic events as we encourage all cases of thrombosis in healthy donors to compared to levels below 1.0 U/ml.18 F1+2 and TAT are be reported. Meanwhile, there has been a suggestion that markers of thrombin generation and have been extensively since most events reported were arterial, anti-platelet agents evaluated in patients with hypercoagulable state. Several should be considered in healthy donors with symptomatic patients with hereditary deficiencies such as antithrombin or severe atherosclerosis or past history of thrombosis.6 Our Effect of G-CSF on hemostasis in allogeneic blood stem cell donors R LeBlanc et al 995 a b 100 3.5 90 3 80 70 2.5 60 2 % 50 U/ml 40 1.5 30 1 20 0.5 10 0 0 A* B* AB

c d 3 3

2.5 2.5

2 2

1.5 1.5 U/ml U/ml

1 1

0.5 0.5

0 0 A B AB

e f 3.5 10 3 8 2.5

6 2

1.5 nmol/l ng/ml 4 1 2 0.5

0 0 AB AB

Figure 1 Graphics present comparisons of levels of hemostasis parameters before and after G-CSF administration in each donor. *A represents levels before G-CSF administration and B, levels after G-CSF. (a) Platelet aggregation test using ADP 3 ␮m at 8 min; (b) vWF:Ag; (c) vWF activity; (d) FVIII:C; (e) TAT; (f) PF1+2.

study has not demonstrated any clear evidence of platelet References aggregation or activation and therefore does not support a role for these agents, especially regarding our findings on 1 Anderlini P, Przepiorka D, Champlin R, Ko¨rbling M. Biologic platelet aggregation. Heparin or other anticoagulants could and clinical effects of granulocyte colony-stimulating factor be of benefit in view of our demonstration of thrombin gen- in normal individuals. Blood 1996; 88: 2819–2825. eration after G-CSF, but we cannot make a recommen- 2 Shimoda K, Okamura S, Harada N et al. Identification of a dation only on the basis of these results. More studies will functional receptor for granulocyte colony-stimulating factor be needed to answer these questions. on platelets. J Clin Invest 1993; 91: 1310–1313. 3 Kanaji T, Okamura T, Nagafuji K et al. Megakaryocytes pro- duce the receptor for granulocyte colony-stimulating factor. Blood 1995; 85: 3359–3360. Acknowledgements 4 Shimoda K, Okamura S, Inaba S et al. Granulocyte colony- stimulating factor and platelet aggregation. Lancet 1993; 341: 633. We are grateful to Amgen Canada for providing us with a grant 5 Avenarius HJ, Freund M, Deinhardt J, Poliwoda H. Effect of to support the laboratory work. recombinant human granulocyte colony-stimulating factor Effect of G-CSF on hemostasis in allogeneic blood stem cell donors R LeBlanc et al 996 (rhG-CSF) on circulating platelets. Ann Hematol 1992; 65: 16 Mannucci PM, Carlsson S, Harris AS. Desmopressin, surgery 6–9. and thrombosis. Thromb Haemost 1994; 71: 154–155. 6 Kuroiwa M, Okamura T, Kanaji T et al. Effects of granulocyte 17 Mannucci PM, Lusher JM. Desmopressin and thrombosis. colony-stimulating factor on the hemostatic system in healthy Lancet 1989; ii: 675–676. volunteers. Int J Hematol 1996; 63: 311–316. 18 Koster T, Blann AD, Brie¨tEet al. Role of clotting factor VIII 7 Rivera J, Zuazu I, Sanchez MJ et al. Effect of G-CSF on hae- in effect of von Willebrand factor on occurrence of deep-vein mostatic system. Thromb Haemost 1994; 71: 804–805. thrombosis. Lancet 1995; 345: 152–155. 8 Kadar JG, Arseniev L, Avenarius HJ et al. Safety of allo- 19 Bauer KA, Goodman TL, Kass BL, Rosenberg RD. Elevated geneic peripheral stem cell mobilisation and collection. Bone factor Xa activity in the blood of asymptomatic patients with Marrow Transplant 1996; 17 (Suppl. 2): 67 (Abstr. 8). congenital antithrombin deficiency. J Clin Invest 1985; 76: 9 Avenarius HJ, Freund M, Kleine HD et al. Granulocyte 826–836. colony-stimulating factor enhances the expression of CD62 on 20 Bauer KA, Broekmans AW, Bertina RM et al. Hemostatic platelets in vivo. Int J Hematol 1993; 58: 189–196. enzyme generation in the blood of patients with hereditary 10 Falanga A, Marchetti M, Oldani E et al. Changes of hemo- protein C deficiency. Blood 1988; 71: 1418–1426. static parameters in healthy donors administered G-CSF for 21 Conway EM, Bauer KA, Barzegar S, Rosenberg RD. Sup- peripheral blood progenitor cells (PBPC) collection. Bone pression of hemostatic system activation by oral anticoagu- Marrow Transplant 1996; 17 (Suppl. 2): 72 (Abstr. 26). lants in the blood of patients with thrombotic diatheses. J Clin 11 Anderlini P, Ko¨rbling M, Dale D et al. Allogeneic blood stem Invest 1987; 80: 1535–1544. cell transplantation: considerations for donors. Blood 1997; 22 Kistler JP, Singer DE, Millenson MM et al. Effect of low- 90: 903–908. 12 Conti JA, Scher HI. Acute arterial thrombosis after escalated- intensity warfarin anticoagulation on level of activity of the dose methotrexate, vinblastine, doxorubicin, and cisplatin hemostatic system in patients with arial fibrillation. Stroke chemotherapy with recombinant granulocyte colony-stimulat- 1993; 24: 1360–1365. ing factor. Cancer 1992; 70: 2699–2702. 23 Knudsen JB, Brodthagen U, Gormsen J et al. Platelet function 13 Kawachi Y, Watanabe A, Uchida T et al. Acute arterial throm- and fibrinolytic activity following distance running. Scand J bosis due to platelet aggregation in a patient receiving gra- Haematol 1982; 29: 425–430. nulocyte colony-stimulating factor. Br J Haematol 1996; 94: 24 Fong JSC and Kaplan BS. Impairment of platelet aggregation 413–416. in hemolytic uremic syndrome: evidence of platelet ‘exhaus- 14 Lindemann A, Rumberger B. Vascular complications in tion’. Blood 1982; 60: 564–570. patients treated with granulocyte colony-stimulating factor (G- 25 Sullivan MHF, Elder MG. Changes in platelet reactivity CSF). Eur J Cancer 1993; 29A: 2338–2339. following aspirin treatment for pre-eclampsia. Br J Obstet 15 Jern C, Eriksson E, Tengborn L et al. Changes of plasma Gynaecol 1993; 100: 542–545. coagulation and fibrinolysis in response to mental stress. Thromb Haemost 1989; 62: 767–771.