Abstract:
The use of pyridoxal-5&#39;-phosphate as an in vitro anticoagulant agent which retains the platelet activity of stored whole blood or stored plasma for more than about six hours is disclosed.

Description:
This is a divisional of co-pending application Ser. No. 7/055,637 filed on May 29, 1987, now U.S. Pat. No. 4,931,002. 
    
    
     BACKGROUND OF THE INVENTION 
     Whole blood is typically stored for very short periods of time prior to use, due to its rapid deterioration due to factors such as clotting, cell breakup and loss of platelet activity. 
     SUMMARY OF THE INVENTION 
     The present invention provides a method for storing whole blood and blood fractions containing platelets (e.g., plasma) so that the loss of platelet activity is reduced. Thus, surprisingly whole blood treated in accord with this invention can be stored for more than six (6) hours, and up to at least about 24 hours, without loss of platelet activity whereas previously whole blood could only be stored for up to 6 hours. In accord with the invention pyridoxal-5&#39;-phosphate is used as an in vitro anticoagulant agent e.g., when drawing blood for storage, fractionation of blood into its useful components, and the like, where retention of platelet activity is desired. 
     Pyridoxal-5&#39;-phosphate is also useful as an additive to whole blood and blood fractions containing platelets, to preserve the activity of platelets for long periods of time. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     It has been discovered that pyridoxal-5&#39;-phosphate (PLP) provides excellent anticoagulant properties during the collection of whole blood. 
     It has further been discovered that the inhibition of blood clotting afforded by PLP lasts for a long period of time (e.g., greater than 24 hr.). 
     Finally, it has been discovered that the inhibition of coagulation by PLP is a reversible process, since washed platelets, when resuspended in PLP-free Tyrodes Buffer, were able to aggregate normally when exposed to ADP and thrombin, indicating that no permanent platelet functional damage had occurred due to exposure to PLP for up to 24 hours at room temperature. 
     PLP is a naturally occurring derivative of vitamin B 6  which has previously been reported as an inhibitor of ADP and thrombin-induced platelet aggregation in both platelet--rich plasma and in a washed platelet system. See for example, Kornicki et al., Amer. J. Physiol., 238: H54-60 (1980) and Kornecki et al., Biochem. Biophys. Res. Commun., 90: 963-968 (1979). 
     While there has been at least one previous study reporting that PLP can act as both an in vitro and in vivo plasma anticoagulant (see, Subbarao et al., Biochem. Pharmacol., 28: 531-534 (1979)), the present inventors are aware of no report which suggest that PLP be used for the collection and/or storage of whole blood and, particularly, no reports that suggest that when using PLP as an anticoagulant for the storage of whole blood, plasma or platelets, there is no loss of platelet activity for at least 24 hours at room temperature. Prolonging of platelet activity can be further increased by lowering the temperature of the material. 
     In fact the findings reported herein were quite surprising to the present inventors, given the well known problems associated with conventional anticoagulants used for blood collection. 
     Typical anticoagulant for blood collection (or its components, e.g., plasma) include acid citrate dextrose (ACD); citrate phosphate dextrose (CPD); citrate phosphate dextrose adenine (CPD-A); sodium citrate and EDTA, which either decrease the pH of the collected blood or chelate essential divalent ions such as Ca 2+ , Mg 2+ , and the like. Heparin, another widely used anticoagulant, has been shown to aggregate platelets in certain cases. Changes in blood PH and/or prolonged chelation of essential ions, is known to cause irreversible damage to the blood cells, especially platelets, due to the disruption of major membrane protein complexes. 
     In contrast to the aforementioned problems, the use of the water-soluble vitamin B 6  derivative, PLP, at neutral blood pH, neither changes the blood pH, nor acts to cause irreversible damage to platelets, thereby avoiding the problems of the prior art compounds. 
     The present invention will be further illustrated with reference to the following examples which aid in the understanding of the present invention, but which are not to be construed as limitations thereof. All percentages reported herein, unless otherwise specified, are percent by weight. All temperatures are expressed in degrees Celsius. 
     EXAMPLE I 
     
         ______________________________________CONCENTRATIONS OF PYRIDOXAL-5&#39;-PHOSPHATETESTED FOR THEIR ANTICOAGULANT ACTIVITYFINAL CONCENTRATION CLOTTINGOF PLP              OF WHOLE BLOOD______________________________________0 PLP               Clot forms in 13 min.0.1 mM PLP*         Clot forms in 18 min.0.3 mM PLP          Clot forms in 18 min.0.5 mM PLP          Clot forms in 18 min.1 mM PLP            Clot forms in 20 min.2 mM PLP            Clot forms in 24 min.4 mM PLP            No clotting5 mM PLP            No clotting______________________________________ *In Example I, a freshly prepared aqueous stock solution of pyridoxal5phosphate was prepared as a 50 mM aqueous solution, pH 7.4, containing 2.23% dextrose. The concentrations of PLP shown were final. 
    
     EXAMPLE II 
     
         ______________________________________EFFECT OF 5.5 mM PYRIDOXAL-5&#39;-PHOSPHATE ONTHE INHIBITION OF CLOTTING OF WHOLE BLOODTime of Incubationof Whole Blood with5.5 mM PLP      Clotting of Whole Blood______________________________________12 hr.          NO24 hr.          NO______________________________________ 
    
     In this Example, whole blood was collected into 50 cc tubes containing 4 ml of an aqueous stock solution of PLP. The final concentration of PLP was 5.5 mM. The tubes were capped and mixed at room temperature for either a 12 hour or a 24 hour period by use of an aliquot mixer. 
     EXAMPLE III 
     
         ______________________________________EFFECT OF THE ADDITION OF PLPPLUS CPDA ON THE CLOTTING OF BLOODCOMPOUNDS TESTED Clotting of Whole Blood?______________________________________PLP (5.5 mM)     NOPLP (5.5 mM) plus CPDA-1*            NOCPDA-1*          NO______________________________________ *In this Example, the CPDA1 solution was the commercially available blood anticoagulant obtained from Fenwal Laboratories, a division of Travenol Laboratories, Deerfield, IL. 
    
     A 63 ml aqueous solution of this USP grade CPDA-1 contained the following: 206 mg citric acid, 1.66 g sodium citrate, 140 mg monobasic sodium phosphate, 2 g dextrose and 17.3 mg adenine. The ratio (v/v) of the CPDA-1 solution to whole blood was 1:7.14. 
     EXAMPLE IV 
     
         ______________________________________INHIBITION OF CLOTTING OF PLASMA BYPYRIDOXAL-5&#39;-PHOSPHATEPlasma Containedthe following:     Clotting in 24 hr______________________________________Control (No PLP)   YesPLP (5.5 mM)       NoPLP (5.5 mM) plus CPDA-1*              NoCPDA-1*            No______________________________________ *In this Example, the CPDA1 solution was the commercially available blood anticoagulant obtained from Fenwal Laboratories, a division of Travenol Laboratories, Deerfield, IL. 
    
     A 63 ml aqueous solution of this USP grade CPDA-1 contained the following: 206 mg citric acid, 1.66 g sodium citrate, 140 mg monobasic sodium phosphate, 2 g dextrose and 17.3 mg adenine. The ratio (v/v) of the CPDA-1 solution to plasma was 1:7.14. 
     EXAMPLE V 
     
         ______________________________________PREPARATION AND TESTING OF PLATELETFUNCTIONAL PROPERTIES IN GEL-FILTEREDPLATELETS OBTAINED FROM FRESH WHOLEBLOOD ANTICOAGULATED IN THE PRESENCE OF5.4 mM PYRIDOXAL-5&#39;-PHOSPHATE         Platelet Aggregation         Parameters*         Initial VelocityAgonists Tested         (LTU/min)      Extent (LTU)______________________________________ADP (49 uM)   96             62ADP (20.8 uM) 84             66Collagen (111 ug/ml)         29             78Thrombin (5.5 U/ml)         120            70A23187 (35 uM)         36             76PAF (24.5 uM) 74             54PAF (1.64 uM) 68             54PAF (326 nM)  92             64PAF (164 nM)  84             64PAF (20.2 nM) 68             56PAF (10.2 nM) 13              9______________________________________ Platelet aggregation was initiated by the addition of the agonists described above. The concentrations shown above were final. *For Examples V-IX, the procedures of Kornecki et al., Science, 226: 1454-1456 (1984) were followed for the preparation of gelfiltered platelets and for aggregation of 0.45 ml aliquots of platelets. 
    
     EXAMPLE VI 
     
         ______________________________________PREPARATION AND TESTING OF THE PLATELETFUNCTIONAL PROPERTIES IN GEL-FILTEREDPLATELETS OBTAINED FROM WHOLE BLOODANTICOAGULATED IN THE PRESENCE OF PLP(5.4 mM) PLUS CPDA-1*         Platelet Aggregation         Parameters**         Initial VelocityAgonist Tested         (LTU/min)      Extent (LTU)______________________________________ADP (49 uM)   44             46ADP (20.8 uM) 54             60Collagen (111 ug/ml)         28             66Thrombin (5.5 U/ml)         76             68A23187 (35 uM)         24             78PAF (24.5 uM) 52             42PAF (1 uM)    27             28PAF (20.2 nM) 14             14______________________________________ *In this Example, the CPDA1 solution was the commercially available blood anticoagulant obtained from Fenwal Laboratories, a division of Travenol Laboratories, Deerfield, IL. A 63 ml aqueous solution of this USP grade CPDA1 contained the following: 206 mg citric acid, 1.66 g sodium citrate, 140 mg monobasic sodium phosphate, 2 g dextrose and 1.3 mg adenine. The ratio (v/v) of the CPDA1 solution to the whole blood was 1:7.14. **See Example V. 
    
     EXAMPLE VII 
     
         ______________________________________PREPARATION AND TESTING OF PLATELETFUNCTIONAL PROPERTIES IN GEL-FILTEREDPLATELETS FROM 24 HR-OLD BLOODANTICOAGULATED IN THE PRESENCE OF 5.4 mM PLP         Platelet Aggregation         Parameters*         Initial VelocityAgonist Tested         (LTU/min)      Extent (LTU)______________________________________ADP (49 uM)   40             44ADP (20.8 uM) 44             54Collagen (111 ug/ml)         19             48Thrombin (5.5 U/ml)         44             48A23187 (35 uM)         32             50PAF (24.5 uM) 36             40PAF (20 nM)   12             18______________________________________ *See Example V. 
    
     EXAMPLE VIII 
     
         ______________________________________RESPONSIVENESS OF GEL-FILTERED PLATELETSPREPARED FROM PLASMA KEPT ANTICOAGULATEDFOR 24 HOURS IN THE PRESENCE OF PLP(5.4 mM): COMPARISON WITH CPDA-1*          Extent of Platelet          Aggregation (LTU)**            Pyridoxal-5&#39;-                       CPDA-1*            Phosphate as                       as anti-Agonists Tested  anticoagulant                       coagulant______________________________________ADP (49 uM)      54         8PAF (20 nM)      26         0Collagen (111 ug/ml)            58         0Thrombin (5.5 U/ml)            58         14______________________________________ *In this Example, the CPDA1 solution was the commercially available blood anticoagulant obtained from Fenwal Laboratories, a division of Travenol Laboratories, Deerfield, IL. A 63 ml aqueous solution of this USP grade CPDA1 contained the following: 206 mg citric acid, 1.66 g sodium citrate, 140 mg monobasic sodium phosphate, 2 g dextrose and 17.3 mg adenine. The ratio (v/v) of the CPDA1 solution to the whole blood was 1:7.14. **See Example V. 
    
     EXAMPLE IX 
     
         ______________________________________THE EFFECT OF THE ADDITION OF PLP TOCPDA-1: RESPONSIVENESS OF GEL-FILTEREDPLATELETS ISOLATED FROM PLASMA STOREDFOR 24 HR IN THE PRESENCE OF PLP(5.4 mM) PLUS CPDA-1*    Extent of Platelet Aggregation (LTU)**                              % Recovery                              by the                              addition                       PLP plus                              of PLP toAgonists Tested      PLP    CPDA-1    CPDA-1 CPDA-1______________________________________ADP (49 uM)      54     8         74     &gt;100PAF (20 nM)      26     0         22       85Collagen   58     0         90     &gt;100(111 ug/ml)Thrombin   58     14        50       86(5.5 U/ml)______________________________________ *In this Example, the CPDA1 solution was the commercially available blood anticoagulant obtained from Fenwal Laboratories, a division of Travenol Laboratories, Deerfield, IL. A 63 ml aqueous solution of this USP grade CPDA1 contained the following: 206 mg citric acid, 1.66 g sodium citrate, 140 mg monobasic sodium phosphate, 2 g dextrose and 17.3 mg adenine. The raito (v/v) of the CPDA1 solution to the plasma was 1:7.14. **See Example V. 
    
     The following are some of the conclusions that can be drawn from the foregoing examples: 
     1. Pyridoxal-5&#39;-phosphate (PLP) at concentrations of about 4 mM and greater, can be used as an effective anticoagulant of whole blood (see Example I), without loss of platelet activity for up to 24 hours. 
     2. Pyridoxal-5&#39;-phosphate, when added to whole blood, can be sued for the purpose of long-term storage of whole blood (at least over a 24-hour period of time) (see Example II). 
     3. Pyridoxal-5&#39;-phosphate can be used as an &#34;additive&#34; combined with conventional anticoagulants used in blood banks, such as CPDA-1 (see Example III), thereby reducing the loss of platelet activity experienced when using such anticoagulants alone. 
     4. Plasmas which were obtained from whole blood collected in tubes containing either a) PLP, b) the standard anticoagulant used in blood banks, CPDA-1, or c) a combination of PLP plus CPDA, did not exhibit any clotting over a 24 hr. incubation period. (see Example IV). 
     5. Platelets, which were isolated from whole blood anticoagulated in the presence of PLP (5.4 mM), exhibited responsiveness to all platelet agonists tested. (see Example V). 
     6. Platelets which were isolated from whole blood anticoagulated in the presence of both PLP (5.4 mM) plus CPDA-1, exhibited responsiveness to all platelet agonists. (see Example VI). 
     7. Platelets, which were isolated from 24 hr. old whole blood anticoagulated in the presence of PLP, exhibited responsiveness to all platelet agonists. (see Example VIl). 
     8. Platelets isolated from platelet-rich plasma, which was kept for 24 hr. with pyridoxal-5&#39;-phosphate, showed responsiveness to all physiological agonists tested. (see Example VIII). 
     9. Platelets isolated from platelet-rich plasma, which was kept for 24 hr. with CPDA-1, showed loss in responsiveness to physiological agonists. (see Example VIII). 
     10. Preservation of platelet responsiveness may be accomplished by the use of PLP as an additive to traditional anticoagulants, such as CPDA-1. (see Example IX). 
     The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention and still be within the scope and spirit of this invention as set forth in the following claims.